Compositions of asymmetric interfering RNA and uses thereof

ABSTRACT

The present invention provides asymmetrical duplex RNA molecules that are capable of effecting sequence-specific gene silencing. The RNA molecule comprises a first strand and a second strand. The first strand is longer than the second strand. The RNA molecule comprises a double-stranded region formed by the first strand and the second strand, and two ends independently selected from the group consisting of 5′-overhang, 3′-overhang, and blunt end. The RNA molecules of the present invention can be used as research tools and/or therapeutics.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of, and claims priorityto and benefit of, co-pending U.S. application Ser. No. 15/081,559,filed Mar. 25, 2016, which, in turn, is a divisional of U.S. applicationSer. No. 12/199,797 filed Aug. 27, 2008, now U.S. Pat. No. 9,328,345 andclaims priority to and the benefit of U.S. provisional patentapplication Ser. No. 60/968,257 filed on Aug. 27, 2007, 61/029,753,filed on Feb. 19, 2008, and 61/038,954 filed on Mar. 24, 2008, theentire contents of which applications are incorporated herein byreference.

BACKGROUND OF THE INVENTION

Gene silencing through RNAi (RNA-interference) by use of small or shortinterfering RNA (siRNA) has emerged as a powerful tool for molecularbiology and holds the potential to be used for therapeutic purposes (deFougerolles et al., 2007; Kim and Rossi, 2007).

RNAi can be theoretically employed to knockdown or silence any diseasegene with specificity and potency. Possible applications of RNAi fortherapeutic purposes are extensive and include genetic, epigenetic, andinfectious diseases, provided that a disease-causing gene is identified.

However, other than the prominent delivery issue, the development ofRNAi-based drugs faces challenges of limited efficacy of siRNA,non-specific effects of siRNA such as interferon-like responses andsense-strand mediated off-target gene silencing, and the prohibitive orhigh cost associated with siRNA synthesis. The gene silencing efficacyby siRNA is limited to about 50% or less for majority of genes inmammalian cells. The manufacture of these molecules is expensive (muchmore expensive than manufacturing antisense deoxynucleotides),inefficient, and requires chemical modification. Finally, theobservation that the extracellular administration of synthetic siRNAscan trigger interferon-like responses has added a significant barrierfor RNAi-based research and RNAi-based therapeutic development.

RNAi can be selectively triggered by synthetic short interfering RNA(siRNAs) or genetic elements encoding short-hairpin RNAs (shRNAs) thatare subsequently cleaved into siRNAs by the ribonuclease III-likeenzyme, Dicer. The biochemical mechanism of gene silencing, not yetfully understood, appears to involve a multi-protein RNA-inducedsilencing complex (RISC). RISC binds, unwinds, and incorporates theanti-sense siRNA strand, which then recognizes and targets perfectlycomplementary mRNAs for cleavage thereby reducing gene expression.Potent gene silencing (1-10 days) is attributable to the catalyticproperties of the RISC complex. The power of RNAi stems from theexquisite specificity that can be achieved. However, off-target RNAieffects are known to occur. Another major side effect is the activationof the interferon-like response by siRNA, which is mediated viadsRNA-dependent protein kinase (PKR) and Toll-like receptors (TLR). Thecapability to induce interferon-like response by siRNA is mainlydetermined by its length. (ibid.)

For gene silencing in mammalian cells, the current art teaches that thestructure of siRNA is a symmetric double stranded RNA with a length of19-21 nucleotides and 3′ overhangs on both ends to be effective inmammalian cells and to avoid cellular innate “anti-viral” responses.(ibid.) It is now well established in the field that this “optimal”structure can still trigger interferon responses, and pose significantchallenges to the development of RNAi-based research and RNAi-basedtherapeutics (Sledz et al., 2003).

There is a need to develop novel approaches to effective RNAi inmammalian cells through a novel design of siRNAs having better efficacyand potency, rapid onset of action, better durability, and a shorterlength of the RNA duplex to avoid non-specific interferon like responseand to reduce the cost of synthesis for research and pharmaceuticaldevelopment of RNAi therapeutics.

The references cited herein are not admitted to be prior art to theclaimed invention.

SUMMARY OF THE INVENTION

The present invention is about a surprising discovery of a novel classof small duplex RNA that can induce potent gene silencing in mammaliancells, which is termed herein asymmetrical interfering RNAs (aiRNA). Thehallmark of this novel class of RNAi-inducers is the length asymmetry ofthe two RNA strands. Such a novel structural design is not onlyfunctionally potent in effecting gene silencing, but offers severaladvantages over the current state-of-art siRNAs. Among the advantages,aiRNA can have RNA duplex structure of much shorter length than thecurrent siRNA, which should reduce the cost of synthesis andabrogate/reduce the length-dependent triggering of nonspecificinterferon-like responses. In addition, the asymmetry of the aiRNAstructure abrogates/reduces the sense-strand mediated off-targeteffects. Furthermore, aiRNA is more efficacious, potent, rapid-onset,and durable than siRNA in inducing gene silencing. AiRNA can be used inall areas that current siRNA or shRNA are being applied or contemplatedto be used, including biology research, R&D research in biotechnologyand pharmaceutical industry, and RNAi-based therapies.

The present invention provides a duplex RNA molecule. The duplex RNAmolecule comprises a first strand with a length from 18-23 nucleotidesand a second strand with a length from 12-17 nucleotides, wherein thesecond strand is substantially complementary to the first strand, andforms a double-stranded region with the first strand, wherein the firststrand has a 3′-overhang from 1-9 nucleotides, and a 5′-overhang from0-8 nucleotides, wherein said duplex RNA molecule is capable ofeffecting selective gene silencing in a eukaryotic cell. In anembodiment, the first strand comprises a sequence being substantiallycomplementary to a target mRNA sequence. In a further embodiment, thefirst strand comprises a sequence being at least 70 percentcomplementary to a target mRNA sequence. In another embodiment, theeukaryotic cell is a mammalian cell or an avian cell.

In an embodiment, at least one nucleotide of the sequence of 5′ overhangis selected from the group consisting of A, U, and dT.

In an embodiment, the GC content of the double stranded region is20%-70%.

In an embodiment, the first strand has a length from 19-22 nucleotides.

In an embodiment, the first strand has a length of 21 nucleotides. In afurther embodiment, the second strand has a length of 14-16 nucleotides.

In an embodiment, the first strand has a length of 21 nucleotides, andthe second strand has a length of 15 nucleotides. In a furtherembodiment, the first strand has a 3′-overhang of 2-4 nucleotides. In aneven further embodiment, the first strand has a 3′-overhang of 3nucleotides.

In an embodiment, the duplex RNA molecule contains at least one modifiednucleotide or its analogue. In a further embodiment, the at least onemodified nucleotide or its analogue is sugar-, backbone-, and/orbase-modified ribonucleotide. In an even further embodiment, thebackbone-modified ribonucleotide has a modification in a phosphodiesterlinkage with another ribonucleotide. In an embodiment, thephosphodiester linkage is modified to include at least one of a nitrogenor sulphur heteroatom. In another embodiment, the nucleotide analogue isa backbone-modified ribonucleotide containing a phosphothioate group.

In an embodiment, the at least one modified nucleotide or its analogueis an unusual base or a modified base. In another embodiment, the atleast one modified nucleotide or its analogue comprises inosine, or atritylated base.

In a further embodiment, the nucleotide analogue is a sugar-modifiedribonucleotide, wherein the 2′-OH group is replaced by a group selectedfrom H, OR, R, halo, SH, SR, NH₂, NHR, NR₂, or CN, wherein each R isindependently C1-C6 alkyl, alkenyl or alkynyl, and halo is F, Cl, Br orI.

In an embodiment, the first strand comprises at least onedeoxynucleotide. In a further embodiment, the at least onedeoxynucleotides are in one or more regions selected from the groupconsisting of 3′-overhang, 5′-overhang, and double-stranded region. Inanother embodiment, the second strand comprises at least onedeoxynucleotide.

The present invention also provides a method of modulating geneexpression in a cell or an organism comprising the steps of contactingsaid cell or organism with the duplex RNA molecule of the inventionunder conditions wherein selective gene silencing can occur, andmediating a selective gene silencing effected by the duplex RNA moleculetowards a target gene or nucleic acid having a sequence portionsubstantially corresponding to the double-stranded RNA. In a furtherembodiment, said contacting step comprises the step of introducing saidduplex RNA molecule into a target cell in culture or in an organism inwhich the selective gene silencing can occur. In an even furtherembodiment, the introducing step is selected from the group consistingof transfection, lipofection, electroporation, infection, injection,oral administration, inhalation, topical and regional administration. Inanother embodiment, the introducing step comprises using apharmaceutically acceptable excipient, carrier, or diluent selected fromthe group consisting of a pharmaceutical carrier, a positive-chargecarrier, a liposome, a protein carrier, a polymer, a nanoparticle, ananoemulsion, a lipid, and a lipoid.

In an embodiment, the modulating method is used for determining thefunction or utility of a gene in a cell or an organism.

In an embodiment, the modulating method is used for treating orpreventing a disease or an undesirable condition.

In an embodiment, the target gene is associated with a disease, apathological condition, or an undesirable condition in a mammal. In afurther embodiment, the target gene is a gene of a pathogenicmicroorganism. In an even further embodiment, the target gene is a viralgene. In another embodiment, the target gene is a tumor-associated gene.In yet another embodiment, the target gene is a gene associated with adisease selected from the group consisting of autoimmune disease,inflammatory diseases, degenerative diseases, infectious diseases,proliferative diseases, metabolic diseases, immune-mediated disorders,allergic diseases, dermatological diseases, malignant diseases,gastrointestinal disorders, respiratory disorders, cardiovasculardisorders, renal disorders, rheumatoid disorders, neurologicaldisorders, endocrine disorders, and aging.

The present invention provides a research reagent. The reagent comprisesthe duplex RNA molecule.

The present invention further provides a kit. The kit comprises a firstRNA strand with a length from 18-23 nucleotides and a second RNA strandwith a length from 12-17 nucleotides, wherein the second strand issubstantially complementary to the first strand, and capable of forminga duplex RNA molecule with the first strand, wherein the duplex RNAmolecule has a 3′-overhang from 1-9 nucleotides, and a 5′-overhang from0-8 nucleotides, wherein said duplex RNA molecule is capable ofeffecting sequence-specific gene silencing in a eukaryotic cell.

The present invention also provides a method of preparing the duplex RNAmolecule. The method comprises the steps of synthesizing the firststrand and the second strand, and combining the synthesized strandsunder conditions, wherein the duplex RNA molecule is formed, which iscapable of effecting sequence-specific gene silencing. In an embodiment,the method further comprises a step of introducing at least one modifiednucleotide or its analogue into the duplex RNA molecule during thesynthesizing step, after the synthesizing and before the combining step,or after the combining step. In another embodiment, the RNA strands arechemically synthesized, or biologically synthesized.

The present invention provides an expression vector. The vectorcomprises a nucleic acid or nucleic acids encoding the duplex RNAmolecule operably linked to at least one expression-control sequence. Inan embodiment, the vector comprises a first nucleic acid encoding thefirst strand operably linked to a first expression-control sequence, anda second nucleic acid encoding the second strand operably linked to asecond expression-control sequence. In another embodiment, the vector isa viral, eukaryotic, or bacterial expression vector.

The present invention also provides a cell. In an embodiment, the cellcomprises the vector. In another embodiment, the cell comprises theduplex RNA molecule. In a further embodiment, the cell is a mammalian,avian, or bacterial cell.

The present invention provides a duplex RNA molecule. The duplex RNAmolecule comprises a first strand and a second strand, wherein the firststrand is longer than the second strand, wherein the second strand issubstantially complementary to the first strand, and forms adouble-stranded region with the first strand, wherein said duplex RNAmolecule is capable of effecting selective gene silencing in aeukaryotic cell. In an embodiment, the two ends of the duplex RNAmolecule are independently selected from the group consisting of3′-overhang from 1-10 nucleotides, 5′-overhang from 0-10 nucleotides,and blunt end. In another embodiment, the first strand is substantiallycomplementary to a target mRNA sequence. In an alternative embodiment,the second strand is substantially complementary to a target mRNAsequence. In an embodiment, the eukaryotic cell is a mammalian cell oran avian cell. In another embodiment, the duplex RNA molecule is anisolated duplex RNA molecule.

In an embodiment, the first strand has a 3′-overhang from 1-8nucleotides and a 5′-overhang from 1-8 nucleotides.

In another embodiment, the first strand has a 3′-overhang from 1-10nucleotides and a blunt end.

In yet another embodiment, the first strand has a 5′-overhang from 1-10nucleotides and a blunt end.

In an alternative embodiment, the RNA duplex has two 5′-overhangs from1-8 nucleotides, or two 3′-overhangs from 1-10 nucleotides.

In an embodiment, the first strand has a length from 12-100 nucleotides,from 12-30 nucleotides from 18-23 nucleotides, from 19-25 nucleotides.In a further embodiment, the first strand has a length of 21nucleotides.

In another embodiment, the second strand has a length from 5-30nucleotides, 12-22 nucleotides, 12-17 nucleotides. In a furtherembodiment, the second strand has a length of 15 nucleotides.

In an embodiment, the first strand has a length from 12-30 nucleotides,and the second strand has a length from 10-29 nucleotides, with theprovision that when the double stranded region is 27 bp, the both endsof the RNA molecule are independently 3′ overhang or 5′ overhang. In afurther embodiment, the first strand has a length from 18-23nucleotides, and the second strand has a length from 12-17 nucleotides.

In another embodiment, the first strand has a length from 19-25nucleotides, and the second strand has a length from 12-17 nucleotides.

In an alternative embodiment, the first strand has a length from 19-25nucleotides, and the second strand has a length from 18-24 nucleotides,

-   -   with the provision that when the first strand is

(SEQ ID NO: 1) 5′-UUCGAAGUAUUCCGCGUACGU (SEQ ID NO: 2)5′-UCGAAGUAUUCCGCGUACGUG or (SEQ ID NO: 3) 5′-CGAAGUAUUCCGCGUACGUGA

the second strand has a length of at most 17 nucleotides, or contains atleast one mismatch with the first strand, or contains at least onemodification.

In an embodiment, the first strand has a length of 21 nucleotides andthe second strand has a length of 12-17 nucleotides, or 14-16nucleotides.

In an embodiment, the first strand is from 1-10 nucleotides longer thanthe second strand.

In an embodiment, the 3′-overhang has a length from 2-6 nucleotides.

In another embodiment, the 5′-overhang has a length from 0-5nucleotides.

In an embodiment, the gene silencing comprises one or two, or all of RNAinterference, modulation of translation, and DNA epigenetic modulations.

In an embodiment, the duplex RNA molecule further comprises a nick in atleast one of said first and second strands.

In another embodiment, the double stranded region comprises a gap of oneor more nucleotides.

In an embodiment, at least one nucleotide of the 5′ overhang is notcomplementary to the target mRNA sequence.

In another embodiment, at least one nucleotide of 5′ overhang isselected from the group consisting of A, U, and dT.

In an embodiment, the duplex RNA molecule is conjugated to an entityselected from the group consisting of peptide, antibody, polymer, lipid,oligonucleotide, cholesterol, and aptamer.

In an embodiment, the RNA molecule further comprises at least oneunmatched or mismatched nucleotide.

In another embodiment, the GC content of the double stranded region is20-70%.

In an embodiment, the 3′-overhang and/or 5′-overhang is stabilizedagainst degradation.

In an embodiment, the duplex RNA molecule contains at least one modifiednucleotide or its analogue. In a further embodiment, the at least onemodified nucleotide or its analogue is sugar-, backbone-, and/orbase-modified ribonucleotide. In a further embodiment, thebackbone-modified ribonucleotide has a modification in a phosphodiesterlinkage with another ribonucleotide. In another embodiment, thephosphodiester linkage is modified to include at least one of a nitrogenor sulphur heteroatom. In yet another embodiment, the nucleotideanalogue is a backbone-modified ribonucleotide containing aphosphothioate group.

In an embodiment, the at least one modified nucleotide or its analoguecomprises a non-natural base or a modified base. In another embodiment,the at least one modified nucleotide or its analogue comprises inosine,or a tritylated base.

In a further embodiment, the nucleotide analogue is a sugar-modifiedribonucleotide, wherein the 2′-OH group is replaced by a group selectedfrom H, OR, R, halo, SH, SR, NH₂, NHR, NR₂, or CN, wherein each R isindependently C1-C6 alkyl, alkenyl or alkynyl, and halo is F, Cl, Br orI.

In an embodiment, the first strand comprises at least onedeoxynucleotide. In a further embodiment, the at least onedeoxynucleotides are in one or more regions selected from the groupconsisting of 3′-overhang, 5′-overhang, and double-stranded regionproximal to the 5′ end of the first strand. In another embodiment, thesecond strand comprises at least one deoxynucleotide.

The present invention also provides a method of modulating geneexpression in a cell or an organism. The method comprises the steps of:contacting said cell or organism with the duplex RNA molecule of claim 1under conditions wherein selective gene silencing can occur, andmediating a selective gene silencing effected by the double-stranded RNAtowards a target nucleic acid having a sequence portion substantiallycorresponding to the double-stranded RNA. In a further embodiment, saidcontacting comprises the step of introducing said duplex RNA moleculeinto a target cell in culture or in an organism in which the selectivegene silencing can occur. In an even further embodiment, the introducingstep is selected from the group consisting of transfection, lipofection,electroporation, infection, injection, oral administration, inhalation,topical and regional administration. In another embodiment, theintroducing step comprises using a pharmaceutically acceptableexcipient, carrier, or diluent selected from the group consisting of apharmaceutical carrier, a positive-charge carrier, a liposome, a proteincarrier, a polymer, a nanoparticle, a nanoemulsion, a lipid, and alipoid. In an embodiment, the modulating method is used for modulatingthe expression of a gene in a cell or an organism.

In another embodiment, the modulating method is used for treating orpreventing a disease or an undesirable condition.

In an embodiment, the target gene is a gene associated with human oranimal diseases. In a further embodiment, the gene is a gene of apathogenic microorganism. In an even further embodiment, the target geneis a viral gene. In another embodiment, the gene is a tumor-associatedgene.

In yet another embodiment, the target gene is a gene associated with adisease selected from the group consisting of autoimmune disease,inflammatory diseases, degenerative diseases, infectious diseases,proliferative diseases, metabolic diseases, immune-mediated disorders,allergic diseases, dermatological diseases, malignant diseases,gastrointestinal disorders, respiratory disorders, cardiovasculardisorders, renal disorders, rheumatoid disorders, neurologicaldisorders, endocrine disorders, and aging.

The modulating method can also be used for studying drug target in vitroor in vivo.

The present invention provides a reagent comprising the duplex RNAmolecule.

The present invention further provides a kit. The kit comprises a firstRNA strand and a second RNA strand, wherein the first strand is longerthan the second strand, wherein the second strand is substantiallycomplementary to the first strand, and capable of forming a duplex RNAmolecule with the first strand, wherein said duplex RNA molecule iscapable of effecting sequence-specific gene silencing in a eukaryoticcell.

The present invention also provides a method of preparing the duplex RNAmolecule of claim 1 comprising the steps of synthesizing the firststrand and the second strand, and combining the synthesized strandsunder conditions, wherein the duplex RNA molecule is formed, which iscapable of effecting sequence-specific gene silencing. In an embodiment,the RNA strands are chemically synthesized, or biologically synthesized.In another embodiment, the first strand and the second strand aresynthesized separately or simultaneously.

In an embodiment, the method further comprises a step of introducing atleast one modified nucleotide or its analogue into the duplex RNAmolecule during the synthesizing step, after the synthesizing and beforethe combining step, or after the combining step.

The present invention further provides a pharmaceutical composition. Thepharmaceutical composition comprises as an active agent at least oneduplex RNA molecule and one or more carriers selected from the groupconsisting of a pharmaceutical carrier, a positive-charge carrier, aliposome, a protein carrier, a polymer, a nanoparticle, a cholesterol, alipid, and a lipoid.

The present invention also provides a method of treatment. The methodcomprises administering an effective amount of the pharmaceuticalcomposition to a subject in need. In an embodiment, the pharmaceuticalcomposition is administered via a route selected from the groupconsisting of iv, sc, inhalation, topical, po, and regionaladministration.

In an embodiment, the first strand comprises a sequence beingsubstantially complimentary to the target mRNA sequence of a geneselected from the group consisting of a developmental gene, an oncogene,a tumor suppresser gene, and an enzyme gene, and a gene for an adhesionmolecule, a cyclin kinase inhibitor, a Wnt family member, a Pax familymember, a Winged helix family member, a Hox family member, acytokine/lymphokine or its receptor, a growth/differentiation factor orits receptor, a neurotransmitter or its receptor, a kinase, a signaltransducer, a viral gene, a gene of an infectious disease, ABLI, BCL1,BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2, ETS1, ETS1, ETV6, FGR,FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCL1,MYCN, NRAS, PIM1, PML, RET, SRC, TAL1, TCL3 and YES, APC, BRCA1, BRCA2,MADH4, MCC, NF1, NF2, RB1, TP53, WT1, an ACP desaturase or hydroxylase,an ADP-glucose pyrophorylase, an ATPase, an alcohol dehydrogenase, anamylase, an amyloglucosidase, a catalase, a cellulase, a cyclooxygenase,a decarboxylase, a dextrinase, a DNA or RNA polymerase, a galactosidase,a glucanase, a glucose oxidase, a GTPase, a helicase, a hemicellulase,an integrase, an invertase, an isomerase, a kinase, a lactase, a lipase,a lipoxygenase, a lysozyme, a pectinesterase, a peroxidase, aphosphatase, a phospholipase, a phosphorylase, a polygalacturonase, aproteinase or peptideases, a pullanase, a recombinase, a reversetranscriptase, a topoisomerase, a xylanase, k-RAS, β-Catenin, Rsk1,PCNA, p70S6K, Survivin, mTOR, PTEN, Parp1, or p21.

In another embodiment, the duplex RNA molecule is selected from thegroup consisting of

aiNbs1 5′-AUGCUGUGUUAACUG (SEQ ID NO: 4) UAGUACGACACAAUUGACGAA-5′(SEQ ID NO: 5) aiEF2 5′-CCUCUUAUGAUGUAU (SEQ ID NO: 6)CCGGGAGAAUACUACAUAUAA-5′ (SEQ ID NO: 7) aiStat3-A5′-AGCAAAGAAUCACAU (SEQ ID NO: 8) CGGUCGUUUCUUAGUGUACAA-5′(SEQ ID NO. 9) aiStat3-B 5′-GAAUCUCAACUUCAG (SEQ ID NO: 10)CGUCUUAGAGUUGAAGUCUAA-5′ (SEQ ID NO: 11) aiPTEN5′-UAAAGGUGAAGAUAU (SEQ ID NO: 12) UCGAUUUCCACUUCUAUAUAA-5′(SEQ ID NO: 13) aip70S6K 5′-UGUUUGAUUUGGAUU (SEQ ID NO: 14)GGCACAAACUAAACCUAAAAA-5′ (SEQ ID NO: 15) aimTOR5′-GAAUUGUCAAGGGAU (SEQ ID NO: 16) CGUCUUAACAGUUCCCUAUAA-5′(SEQ ID NO: 17) aiRsk1 5′-AAUUGGAACACAGUU (SEQ ID NO: 18)CCUUUAACCUUGUGUCAAAAA-5′ (SEQ ID NO: 19) aiPCNA5′-AGAUGCUGUUGUAAU (SEQ ID NO: 20) ACCUCUACGACAACAUUAAAA-5′(SEQ ID NO: 21) aiParp1 5′-GCGAAGAAGAAAUCU (SEQ ID NO: 22)CACCGCUUCUUCUUUAGAUAA-5′ (SEQ ID NO: 23) aiSurvivin5′-AGGAGAUCAACAUUU (SEQ ID NO: 24) dTdTUUCCUCUAGUUGUAAAAGU-5′(SEQ ID NO: 25) aiNQO1 5′-GCAGACCUUGUGAUA (SEQ ID NO: 26)CGGCGUCUGGAACACUAUAAA-5′ (SEQ ID NO: 27) aip21-A5′-CCCGCUCUACAUCUU (SEQ ID NO: 28) UCCGGGCGAGAUGUAGAAGAA-5′(SEQ ID NO: 29) aip21-B 5′-GGCGGUUGAAUGAGA (SEQ ID NO: 30)GAUCCGCCAACUUACUCUCAA-5′ (SEQ ID NO: 31) aik-Ras5′-GGAGCUGUUGGCGUA (SEQ ID NO: 32) CAACCUCGACAACCGCAUCAA-5′(SEQ ID NO: 33) aiβ-catenin-1 5′-GCUGAUAUUGAUGGA (SEQ ID NO: 34)CAUCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 39) aiβ-catenin-25′-GAUAUUGAUGGACUU (SEQ ID NO: 36) CAUCGACUAUAACUACCUGAA-5′(SEQ ID NO: 39) aiβ-catenin-4 5′-CUGAUAUUGAUGGAC (SEQ ID NO: 38)CAUCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 39) aiβ-catenin-55′-AGCUGAUAUUGAUGG (SEQ ID NO: 40) CAUCGACUAUAACUACCUGAA-5′(SEQ ID NO: 39) aiβ-catenin-8 5′-UAGCUGAUAUUGAUG (SEQ ID NO: 41)CAUCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 39) aiβ-catenin-95′-UGAUAUUGAUGGACU (SEQ ID NO: 42) CAUCGACUAUAACUACCUGAA-5′(SEQ ID NO: 39) aiβ-catenin-10 5′-GCUGAUAUUGAUGGA (SEQ ID NO: 34)UCAUCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 43) aiβ-catenin-115′-GCUGAUAUUGAUGGA (SEQ ID NO: 34) CAUCGACUAUAACUACCUGAAA-5′(SEQ ID NO: 35) aiβ-catenin-18 5′-GCUGAUAUUGAUGGA (SEQ ID NO: 34)AUCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 37) aiβ-catenin-345′-GCUGAUAUUGAUGGAC (SEQ ID NO: 44) CAUCGACUAUAACUACCUGAA-5′(SEQ ID NO: 39) aiβ-catenin-35 5′-AGCUGAUAUUGAUGGA (SEQ ID NO: 45)CAUCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 39) aiβ-catenin-365′-AGCUGAUAUUGAUGGAC (SEQ ID NO: 46) CAUCGACUAUAACUACCUGAA-5′(SEQ ID NO: 39) aiβ-catenin-37 5′-AGCUGAUAUUGAUGGACU (SEQ ID NO: 47)CAUCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 39) aiβ-catenin-385′-UAGCUGAUAUUGAUGGAC (SEQ ID NO: 48) CAUCGACUAUAACUACCUGAA-5′(SEQ ID NO: 39) aiβ-catenin-39 5′-GCUGAUAUUGAUGGACUU (SEQ ID NO: 49)CAUCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 39) aiβ-catenin-405′-AGCUGAUAUUGAUGGACUU (SEQ ID NO: 50) CAUCGACUAUAACUACCUGAA-5′(SEQ ID NO: 39) aiβ-catenin-42 5′-UAGCUGAUAUUGAUGGACU (SEQ ID NO: 51)CAUCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 39) aiβ-catenin-435′-UAGCUGAUAUUGAUGGACUU (SEQ ID NO: 52) CAUCGACUAUAACUACCUGAA-5′(SEQ ID NO: 39) aiβ-catenin-44 5′-GUAGCUGAUAUUGAUGGACU (SEQ ID NO: 53)CAUCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 39) aiβ-catenin-455′-GCUGAUAUUGAAGGA (SEQ ID NO: 54) CAUCGACUAUAACUACCUGAA-5′(SEQ ID NO: 39) aiβ-catenin-46 5′-GCUGAUAUUGAUGGA (SEQ ID NO: 34)CAUCGACUAUAACUACCUGUC-5′ (SEQ ID NO: 55) aiβ-catenin-475′-GCUGAUAUUGAUGGA (SEQ ID NO: 34) CAUCGACUAUAACUACCUgaa-5′(SEQ ID NO: 56) aiβ-catenin-48 5′-GCUGAUAUUGAUGGA (SEQ ID NO: 34)catCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 57) aiβ-catenin-525′-gCUGAUAUUGAUGGA (SEQ ID NO: 58) CAUCGACUAUAACUACCUGAA-5′(SEQ ID NO: 39) aiβ-catenin-53 5′-GCUGAUAUUGAUGGa (SEQ ID NO: 59)CAUCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 39) aiβ-catenin-575′-gCUGAUAUUGAUGGA (SEQ ID NO: 58) catCGACUAUAACUACCUGAA-5′(SEQ ID NO: 57) aiβ-catenin-59 5′-GCUGAUAUUGAUGGa (SEQ ID NO: 59) andcatCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 57) aiβ-catenin-625′-UAGCUGAUAUUGAUGGACU (SEQ ID NO: 51) CAUCGACUAUAACUACCUgaa-5′(SEQ ID NO: 56)

Wherein A, U, G, and C are nucleotides, while a, t, g, and c aredeoxynucleotides.

The present invention provides a method of modifying a first duplex RNAmolecule with an antisense strand and a sense strand that form adouble-stranded region. The method comprises, shortening the length ofthe sense strand so that the antisense strand has a 3′-overhang from 1-8nucleotides and a 5′-overhang from 0-8 nucleotides, and forming a secondduplex RNA molecule; wherein at least one property of the first duplexRNA molecule is improved. In an embodiment, the property is selectedfrom the group consisting of size, efficacy, potency, the speed ofonset, durability, synthesis cost, off-target effects, interferonresponse, and delivery. In another embodiment, the method furthercomprises combining the antisense strand and the shortened sense strandunder conditions, wherein the second duplex RNA molecule is formed. In afurther embodiment, the first duplex RNA molecule is a siRNA, ordicer-substrate siRNA, or a siRNA precursor.

The present invention provides an expression vector. The vectorcomprises a nucleic acid or nucleic acids encoding the duplex RNAmolecule of claim 1 operably linked to at least one expression-controlsequence. In an embodiment, the expression vector comprises a firstnucleic acid encoding the first strand operably linked to a firstexpression-control sequence, and a second nucleic acid encoding thesecond strand operably linked to a second expression-control sequence.In another embodiment, the expression vector is a viral, eukaryoticexpression vector, or bacterial expression vector.

The present invention also provides a cell. In an embodiment, the cellcomprises the expression, vector. In another embodiment, the cellcomprises the duplex RNA molecule. In yet another embodiment, the cellis a mammalian cell, avian cell, or bacterial cell.

Other features and advantages of the present invention are apparent fromthe additional descriptions provided herein including the differentexamples. The provided examples illustrate different components andmethodology useful in practicing the present invention. The examples donot limit the claimed invention. Based on the present disclosure theskilled artisan can identify and employ other components and methodologyuseful for practicing the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the structure of a duplex RNA molecule that has both a3′-overhang and a 5′-overhang on the first strand. FIG. 1B shows thestructure of a duplex RNA molecule that has both a 3′-overhang and a5′-overhang on the first strand, and a nick in the duplex. FIG. 1C showsthe structure of a duplex RNA molecule that has both a 3′-overhang and a5′-overhang on the first strand, and a gap in the second strand.

FIG. 2A shows the structure of a duplex RNA molecule that has a bluntend, and a 5′-overhang on the first strand. FIG. 2B shows the structureof a duplex RNA molecule that has a blunt end, and a 3′-overhang on thefirst strand. FIG. 2C shows the structure of a duplex RNA molecule thathas 3′-overhangs on both ends of the duplex and that the first strand islonger than the second strand. FIG. 2D shows the structure of a duplexRNA molecule that has 5′-overhangs on both ends of the duplex and thatthe first strand is longer than the second strand.

FIGS. 3A and 3B show the induction of gene silencing of β-catenin byaiRNA (asymmetric interfering RNAs). FIG. 3A shows the confirmation ofthe oligos. After annealing, the oligos were confirmed by 20%polyacrylamide gel. Lane 1, 21nt/21nt; lane 2, 12nt (a)/21nt; lane 3,12nt (b)/21nt; lane 4, 13nt/13nt; lane 5, 13nt/21nt; lane 6, 14nt/14nt;lane 7, 14nt(a)/21nt; lane 8, 14nt(b)/21nt; lane 9, 15nt/15nt; lane 10,15nt/21nt. FIG. 3B shows the effects of the oligos in gene silencing.HeLa cells were plated at 200,000 cells/well into a 6 well cultureplate. 24 hours later they were transfected with scramble siRNA (lane1), 21-bp siRNA targeted E2F1 (lane 2, as a control for specificity) or21-bp siRNA targeted beta-catenin (lane 3, as a positive control), orthe same concentration of aiRNA of different length mix: 12nt(a)/21nt(lane 4); 12nt (b)/21nt (lane 5); 13nt/21nt (lane 6); 14nt (a)/21nt(lane 7); 14nt (b)/21nt (lane 8); 15nt/21nt (lane 9). Cells wereharvested 48 hours after transfection. Expression of β-catenin wasdetermined by Western blot. E2F1 and actin are used as controls.

FIG. 4: shows the structure-activity relationship of aiRNA oligos, withor without base substitutions, in mediating gene silencing. Hela cellswere transfected with the indicated aiRNA. Cells were harvested andlysates generated at 48 hours post transfection. Western blots wereperformed to detect levels of b-catenin and actin. si stands forb-catenin siRNA oligonucleotide. The numerical labeling above each lanecorresponds to the aiRNA oligos in Table 3.

FIG. 5: shows the structure-activity relationship of aiRNA oligos, withor without base substitutions, in mediating gene silencing. Hela cellswere transfected with the indicated aiRNA. Cells were harvested andlysates generated at 48 hours post transfection. Western blots wereperformed to detect levels of b-catenin and actin. si stands forb-catenin siRNA oligonucleotide. The numerical labeling above each lanecorresponds to the aiRNA oligos in Table 3.

FIGS. 6A-6F show the analysis of the mechanism of gene silencingtriggered by aiRNA. FIG. 6A shows the northern blot analysis ofb-catenin mRNA levels in cells transfected with aiRNA or siRNA for theindicated number of days. FIG. 6B shows the schematic of 5′-RACE-PCR forb-catenin showing cleavage of mRNA and expected PCR product. FIG. 6Cshows b-catenin cleavage products mediated by aiRNA were amplified by5′-RACE-PCR from cells transfected with aiRNA for 4 or 8 hours. FIG. 6Dshows the schematic of b-catenin mRNA cleavage site confirmed bysequencing the 5′-RACE-PCR fragment (SEQ ID NOs:34 and 39). FIG. 6Eshows differential RISC loading efficiency of aiRNA and siRNA. aiRNA orsiRNA duplexes were transfected into Hela cells 48 hours aftertransfection with pCMV-Ago2. Ago2 was immunoprecipitated at theindicated time points following aiRNA or siRNA transfection, andnorthern blot analysis was performed to determine levels of Ago2/RISCassociated small RNAs. Levels of Ago2 (shown below) were determined bywestern blot following IP. FIG. 6F shows the effects of knocking downAgo2 or Dicer on gene silencing activity of aiRNA and siRNA. Cells weretransfected with scramble siRNA (siCon), or siRNA targeting Ago2(siAgo2), or Dicer (siDicer) 24 hours prior to transfection withscramble aiRNA (Con) or aiRNA targeting Stat3 (ai). Cells were harvestedand western blot analysis was performed at 48 hours following aiStat3transfection.

FIGS. 7A and 7B show the advantages of incorporation of aiRNA into RISCcompared to siRNA. FIG. 7A shows that aiRNA enters RISC with betterefficiency than siRNA. Cells transfected with Ago2 expression plasmidwere transfected with aiRNA or siRNA for the indicated times. Followingcell lysis, Ago2 was immunoprecipitaed, RNA extracted from theimmunoprecipitate, and separated on a 15% acrylamide gel. Followingtransfer, the membrane was hybridized to a probe to detect the 21 merantisense strand of the aiRNA or siRNA. IgG control lane shows lack ofsignal compared to Ago2 immunoprecipitate. FIG. 7B shows that the sensestrand of aiRNA does not stay in RISC. Membrane from FIG. 7A wasstripped and re-probed to detect the sense strand of the transfectedoligo. Cartoons in FIGS. 7A and 7B illustrate the position of the sensestrand (upper strand), the antisense strand (lower strand), or theduplex on the membrane.

FIGS. 8A and 8B show that the mechanism of RISC loading by aiRNA. FIG.8A shows the immunoprecipitation analysis of the interaction betweendifferent strands of aiRNA or siRNA and Ago2. Hela S-10 lysatecontaining overexpressed Ago2 was incubated with the indicated aiRNA orsiRNA duplex containing ³²P end labeled sense or antisense strands. The(*) marks the location of the label. Following Ago2 immunoprecipitation,the RNA was isolated and separated on a 15% acrylamide gel and exposedto film. The Ago2-associated RNAs are shown in the pellet fraction,while the non-Ago2 bound RNAs remain in the supernatant (Sup). FIG. 8Bshows the role of sense strand cleavage in aiRNA activity. Cells weretransfected with aiRNA or aiRNA with sense strand 2′-O-methyl atposition 8 (predicted Ago2 cleavage site) or position 9 as a control.RNA was collected at 4 hours post transfection and qRT-PCR performed todetermine relative levels of b-catenin mRNA remaining.

FIGS. 9A-9C show the aiRNA and siRNA competition analysis. FIG. 9Aillustrates the siRNA and aiRNA duplex containing ³²P end labeledantisense strands. The (*) marks the location of the label. FIG. 9Bshows that the cold aiRNA does not compete with labeled siRNA for Ago2.Hela S-10 lysate containing overexpressed Ago2 was incubated with the³²P end labeled siRNA and cold aiRNA or siRNA duplex prior to Ago2immunoprecipitation. RNA was then isolated and analyzed on 15%acrylamide gel. FIG. 9C shows that the cold siRNA does not compete withlabeled aiRNA for Ago2. The same S-10 lysate used in B was incubatedwith the ³²P end labeled aiRNA and cold aiRNA or siRNA duplex prior toAgo2 immunoprecipitation. RNA was then isolated and analyzed on 15%acrylamide gel.

FIG. 10 illustrates the model of aiRNA and siRNA showing observeddifferences in RISC loading and generation of mature RISC.

FIGS. 11A-11D show asymmetric RNA duplexes of 14-15 bp with antisenseoverhangs (aiRNA) induced potent, efficacious, rapid, and durable genesilencing. FIG. 11A shows the Diagram showing sequence and design ofsiRNA (SEQ ID NOs:71 and 107) and aiRNA targeting β-cantenin (Table 3).FIG. 11B shows the induction of gene silencing by aiRNA of variouslengths. ß-catenin protein levels were analysed by western blot in cellstransfected with indicated aiRNA for 48 hours. FIG. 11C shows that aiRNAis more potent and efficacious than siRNA in inducing ß-catenin proteindepletion. Hela cells were transfected with aiRNA or siRNA targetingß-catenin at the indicated concentrations. At 48 hourspost-transfection, cell lysates were made and western blot analysis wasdone. FIG. 11D shows that the aiRNA is more efficacious, rapid, anddurable than siRNA in reducing β-cantenin RNA levels. Cells weretransfected with 10 nM 15 bp aiRNA or 21-mer siRNA for the indicatednumber of days before northern blot analysis.

FIGS. 12A-12D show that aiRNA mediates rapid and potent silencing. FIG.12A shows the sequence and structure of aiRNA (SEQ ID NOs:34 and 39) andsiRNA used to target b-catenin (SEQ ID NOs:71 and 107). FIG. 12B showsRT-PCR of b-catenin mRNA levels from cells transfected with controlaiRNA or aiRNA targeting b-catenin. RNA was collected at the indicatedtimes post transfection. FIG. 12C shows the quantitative real-timeRT-PCR of b-catenin mRNA levels in cells transfected with control,aiRNA, or siRNA for the indicated number of hours. FIG. 12D shows thewestern blot analysis of b-catenin protein levels in cells transfectedwith control, aiRNA, or siRNA for the indicated times.

FIGS. 13A-13D show the comparison of aiRNA with siRNA in gene silencingefficacy and durability against multiple targets. Hela cells weretransfected with scramble siRNA (c), aiRNA (ai), or siRNA (si) targeting(FIG. 13A) b-catenin at 10 nM, (FIG. 13B) Stat3, (FIG. 13C) EF2, or(FIG. 13D) NQO1 at 20 nM. RNA and protein was purified at the indicatedtime points and analyzed for mRNA levels by quantitative real timepolymerase chain reaction (qRT-PCR) and protein levels by western blot.qRT-PCR data is normalized to siCon transfected cells.

FIGS. 14A-14D show aiRNA mediated gene silencing is effective againstvarious genes in multiple cell lines. FIG. 14A shows aiRNA duplex ismore efficacious than siRNA in targeting b-catenin in differentmammalian cell lines. FIG. 14B shows the western blot analysis of Nbs1,Survivin, Parp1, p21 from cells transfected with 20 nM of the indicatedaiRNA or siRNA for 48 hours. FIG. 14C shows the western blot analysis ofRsk1, PCNA, p70S6K, mTOR, and PTEN from cells transfected with 20 nM ofthe indicated aiRNA or siRNA for 48 hours. FIG. 14D shows theallele-specific gene silencing of k-Ras by aiRNA. aiRNA targetingwildtype k-Ras was tested for silencing of k-Ras in both k-Ras wildtype(DLD1) and k-Ras mutant (SW480) cell lines by western blot analysis.

FIGS. 15A-15F show the lack of off-target gene silencing bysense-strand, immunostimulation, and serum stability of aiRNAs. FIG. 15Ashows RT-PCR analysis of the expression of interferon inducible genes inPBMC mock treated or incubated with b-catenin siRNA or aiRNA duplex for16 hours. FIG. 15B shows RT-PCR analysis of the expression of interferoninducible genes in Hela cells mock transfected or transfected with EF2or Survivin aiRNA or siRNA for 24 hours. FIG. 15C shows the microarrayanalysis for changes in the expression of known interferon responserelated genes. Total RNA isolated from aiRNA and siRNA transfected Helacells was analyzed by microarray. FIG. 15D shows that no sense-strandmediated off-target gene silencing is detected for aiRNA. Cells wereco-transfected with aiRNA or siRNA and either a plasmid expressing Stat3(sense RNA) or a plasmid expressing antisense Stat3 (antisense RNA).Cells were harvested and RNA collected at 24 hours post transfection andrelative levels of Stat3 sense or antisense RNA were determined byquantitative real time PCR or RT-PCR (inserts). FIG. 15E shows theStability of aiRNA and siRNA duplexes in human serum. aiRNA and siRNAduplexes were incubated in 10% human serum at 37° C. for the indicatedamount of time prior to gel electrophoresis. Duplex remaining (% ofcontrol) is indicated. FIG. 15F illustrates the proposed model for genespecific silencing mediated by the aiRNA duplex.

FIG. 16 shows the potent Anti-Tumor Activity of aiRNA against b-cateninin SW480 human colon xenografted mouse model. Immunosurpressed mice withestablished subcutaneous SW480 human colon cancer were givenintravenously (iv) with 0.6 nmol PEI-complexed β-cantenin siRNAs,PEI-complexed β-cantenin aiRNAs or a PEI-complexed unrelated siRNA asnegative control daily. Tumor size was evaluated periodically duringtreatment. Each point represents the mean±SEM of six tumors.

FIG. 17 shows the potent Anti-Tumor Activity of aiRNA against b-cateninin HT29 human colon xenografted mouse model. Immunosurpressed mice withestablished subcutaneous HT29 human colon cancer were givenintravenously (iv) with 0.6 nmol PEI-complexed β-cantenin siRNAs,PEI-complexed β-cantenin aiRNAs or a PEI-complexed unrelated siRNA asnegative control every other day. Tumor size was evaluated periodicallyduring treatment. Each point represents the mean±SEM of five tumors.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an asymmetric duplex RNA molecule thatis capable of effecting selective gene silencing in a eukaryotic cell.In an embodiment, the duplex RNA molecule comprises a first strand and asecond strand. The first strand is longer than the second strand. Thesecond strand is substantially complementary to the first strand, andforms a double-stranded region with the first strand.

In an embodiment, the duplex RNA molecule has a 3′-overhang from 1-8nucleotides and a 5′-overhang from 1-8 nucleotides, a 3′-overhang from1-10 nucleotides and a blunt end, or a 5′-overhang from 1-10 nucleotidesand a blunt end. In another embodiment, the duplex RNA molecule has two5′-overhangs from 1-8 nucleotides or two 3′-overhangs from 1-10nucleotides. In a further embodiment, the first strand has a 3′-overhangfrom 1-8 nucleotides and a 5′-overhang from 1-8 nucleotides. In an evenfurther embodiment, the duplex RNA molecule is an isolated duplex RNAmolecule.

In an embodiment, the first strand has a 3′-overhang from 1-10nucleotides, and a 5′-overhang from 1-10 nucleotides or a 5′-blunt end.In another embodiment, the first strand has a 3′-overhang from 1-10nucleotides, and a 5′-overhang from 1-10 nucleotides. In an alternativeembodiment, the first strand has a 3′-overhang from 1-10 nucleotides,and a 5′-blunt end.

In an embodiment, the first strand has a length from 5-100 nucleotides,from 12-30 nucleotides, from 15-28 nucleotides, from 18-27 nucleotides,from 19-23 nucleotides from 20-22 nucleotides, or 21 nucleotides.

In another embodiment, the second strand has a length from 3-30nucleotides, from 12-26 nucleotides, from 13-20 nucleotides, from 14-23nucleotides, 14 or 15 nucleotides.

In an embodiment, the first strand has a length from 5-100 nucleotides,and the second strand has a length from 3-30 nucleotides; or the firststrand has a length from 10-30 nucleotides, and the second strand has alength from 3-29 nucleotides; or the first strand has a length from12-30 nucleotides and the second strand has a length from 10-26nucleotides; or the first strand has a length from 15-28 nucleotides andthe second strand has a length from 12-26 nucleotides; or the firststrand has a length from 19-27 nucleotides and the second strand has alength from 14-23 nucleotides; or the first strand has a length from20-22 nucleotides and the second strand has a length from 14-15nucleotides. In a further embodiment, the first strand has a length of21 nucleotides and the second strand has a length of 13-20 nucleotides,14-19 nucleotides, 14-17 nucleotides, 14 or 15 nucleotides.

In an embodiment, the first strand is at least 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 nucleotides longer than the second strand.

In an embodiment, the duplex RNA molecule further comprises 1-10unmatched or mismatched nucleotides. In a further embodiment, theunmatched or mismatched nucleotides are at or near the 3′ recessed end.In an alternative embodiment, the unmatched or mismatched nucleotidesare at or near the 5′ recessed end. In an alternative embodiment, theunmatched or mismatched nucleotides are at the double-stranded region.In another embodiment, the unmatched or mismatched nucleotide sequencehas a length from 1-5 nucleotides. In an even further embodiment, theunmatched or mismatched nucleotides form a loop structure.

In an embodiment, the first strand or the second strand contains atleast one nick, or formed by two nucleotide fragments.

In an embodiment, the gene silencing is achieved through one or two, orall of RNA interference, modulation of translation, and DNA epigeneticmodulations.

As used in the specification and claims, the singular form “a”, “an”,and “the” include plural references unless the context clearly dictateotherwise. For example, the term “a cell” includes a plurality of cellsincluding mixtures thereof.

As used herein, a “double stranded RNA,” a “duplex RNA” or a “RNAduplex” refers to an RNA of two strands and with at least onedouble-stranded region, and includes RNA molecules that have at leastone gap, nick, bulge, and/or bubble either within a double-strandedregion or between two neighboring double-stranded regions. If one strandhas a gap or a single-stranded region of unmatched nucleotides betweentwo double-stranded regions, that strand is considered as havingmultiple fragments. A double-stranded RNA as used here can have terminaloverhangs on either end or both ends. In some embodiments, the twostrands of the duplex RNA can be linked through certain chemical linker.

As used herein, an “antisense strand” refers to an RNA strand that hassubstantial sequence complementarity against a target messenger RNA. Anantisense strand can be part of an siRNA molecule, part of amiRNA/miRNA* duplex, or a single-strand mature miRNA.

The term “isolated” or “purified” as used herein refers to a materialthat is substantially or essentially free from components that normallyaccompany it in its native state. Purity and homogeneity are typicallydetermined using analytical chemistry techniques such as polyacrylamidegel electrophoresis or high performance liquid chromatography.

As used herein, “modulating” and its grammatical equivalents refer toeither increasing or decreasing (e.g., silencing), in other words,either up-regulating or down-regulating. As used herein, “genesilencing” refers to reduction of gene expression, and may refer to areduction of gene expression about 20%, 30%, 40%, 50%, 60%, 70%, 80%,90% or 95% of the targeted gene.

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to humans, non-human primates,rodents, and the like, which is to be the recipient of a particulartreatment. Typically, the terms “subject” and “patient” are usedinterchangeably herein in reference to a human subject.

Terms such as “treating” or “treatment” or “to treat” or “alleviating”or “to alleviate” as used herein refer to both (1) therapeutic measuresthat cure, slow down, lessen symptoms of, and/or halt progression of adiagnosed pathologic condition or disorder and (2) prophylactic orpreventative measures that prevent or slow the development of a targetedpathologic condition or disorder. Thus those in need of treatmentinclude those already with the disorder; those prone to have thedisorder; and those in whom the disorder is to be prevented. A subjectis successfully “treated” according to the methods of the presentinvention if the patient shows one or more of the following: a reductionin the number of or complete absence of cancer cells; a reduction in thetumor size; inhibition of or an absence of cancer cell infiltration intoperipheral organs including the spread of cancer into soft tissue andbone; inhibition of or an absence of tumor metastasis; inhibition or anabsence of tumor growth; relief of one or more symptoms associated withthe specific cancer; reduced morbidity and mortality; and improvement inquality of life.

As used herein, the terms “inhibiting”, “to inhibit” and theirgrammatical equivalents, when used in the context of a bioactivity,refer to a down-regulation of the bioactivity, which may reduce oreliminate the targeted function, such as the production of a protein orthe phosphorylation of a molecule. In particular embodiments, inhibitionmay refer to a reduction of about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%or 95% of the targeted activity. When used in the context of a disorderor disease, the terms refer to success at preventing the onset ofsymptoms, alleviating symptoms, or eliminating the disease, condition ordisorder.

As used herein, the term “substantially complementary” refers tocomplementarity in a base-paired, double-stranded region between twonucleic acids and not any single-stranded region such as a terminaloverhang or a gap region between two double-stranded regions. Thecomplementarity does not need to be perfect; there may be any number ofbase pair mismatches, for example, between the two nucleic acids.However, if the number of mismatches is so great that no hybridizationcan occur under even the least stringent hybridization conditions, thesequence is not a substantially complementary sequence. When twosequences are referred to as “substantially complementary” herein, itmeans that the sequences are sufficiently complementary to each other tohybridize under the selected reaction conditions. The relationship ofnucleic acid complementarity and stringency of hybridization sufficientto achieve specificity is well known in the art. Two substantiallycomplementary strands can be, for example, perfectly complementary orcan contain from 1 to many mismatches so long as the hybridizationconditions are sufficient to allow, for example discrimination between apairing sequence and a non-pairing sequence. Accordingly, substantiallycomplementary sequences can refer to sequences with base-paircomplementarity of 100, 95, 90, 80, 75, 70, 60, 50 percent or less, orany number in between, in a double-stranded region.

As used herein, antagomirs are miRNA inhibitors, and can be used insilencing endogenous miRNAs. As used herein, mimetics or mimics aremiRNA agonists, and can be used to replace endogenous miRNAs asfunctional equivalents and thereby upregulating pathways affected bysuch endogenous miRNAs.

1. RNA Interference

RNA interference (abbreviated as RNAi) is a cellular process for thetargeted destruction of single-stranded RNA (ssRNA) induced bydouble-stranded RNA (dsRNA). The ssRNA is gene transcript such as amessenger RNA (mRNA). RNAi is a form of post-transcriptional genesilencing in which the dsRNA can specifically interfere with theexpression of genes with sequences that are complementary to the dsRNA.The antisense RNA strand of the dsRNA targets a complementary genetranscript such as a messenger RNA (mRNA) for cleavage by aribonuclease.

In RNAi process, long dsRNA is processed by a ribonuclease protein Dicerto short forms called small interfering RNA (siRNA). The siRNA isseparated into guide (or antisense) strand and passenger (or sense)strand. The guide strand is integrated intoRNA-induced-silencing-complex (RISC), which is a ribonuclease-containingmulti-protein complex. The complex then specifically targetscomplementary gene transcripts for destruction.

RNAi has been shown to be a common cellular process in many eukaryotes.RISC, as well as Dicer, is conserved across the eukaryotic domain. RNAiis believed to play a role in the immune response to virus and otherforeign genetic material.

Small interfering RNAs (siRNAs) are a class of short double-stranded RNA(dsRNA) molecules that play a variety of roles in biology. Most notably,it is involved in the RNA interference (RNAi) pathway where the siRNAinterferes with the expression of a specific gene. In addition, siRNAsalso play roles in the processes such as an antiviral mechanism orshaping the chromatin structure of a genome. In an embodiment, siRNA hasa short (19-21 nt) double-strand RNA (dsRNA) region with 2-3 nucleotide3′ overhangs with 5′-phosphate and 3′-hydroxyl termini.

MicroRNAs (miRNAs) are a class of endogenous, single or double-stranded,about 22 nucleotide-long RNA molecules that regulate as much as 30% ofmammalian genes (Czech, 2006; Eulalio et al., 2008; Mack, 2007). MiRNArepresses protein production by blocking translation or causingtranscript degradation. A miRNA may target 250-500 different mRNAs.MiRNA is the product of the Dicer digestion of pre-miRNA, which is theproduct of primary miRNA (pri-miRNA).

As used herein, antagomirs are miRNA inhibitors, and can be used in thesilencing of endogenous miRNAs. As used herein, mimetics are miRNAagonists, and can be used to replace the miRNAs and downregulate mRNAs.

Dicer is a member of RNase III ribonuclease family. Dicer cleaves longdouble-stranded RNA (dsRNA), pre-microRNA (miRNA), and short hairpin RNA(shRNA) into short double-stranded RNA fragments called smallinterfering RNA (siRNA) about 20-25 nucleotides long, usually with atwo-base overhang on the 3′ end. Dicer catalyzes the first step in theRNA interference pathway and initiates formation of the RNA-inducedsilencing complex (RISC), whose catalytic component argonaute is anendonuclease capable of degrading messenger RNA (mRNA) whose sequence iscomplementary to that of the siRNA guide strand.

As used herein, an effective siRNA sequence is a siRNA that is effectivein triggering RNAi to degrade the transcripts of a target gene. Notevery siRNA complementary to the target gene is effective in triggeringRNAi to degrade the transcripts of the gene. Indeed, time-consumingscreening is usually necessary to identify an effective siRNA sequence.In an embodiment, the effective siRNA sequence is capable of reducingthe expression of the target gene by more than 90%, more than 80%, morethan 70%, more than 60%, more than 50%, more than 40%, or more than 30%.

The present invention provides a novel structural scaffold calledasymmetric interfering RNA (aiRNA) that can be used to effect siRNA-likeresults, and also to modulate miRNA pathway activities (described indetail in co-owned PCT and U.S. applications filed on the same day asthe present application under the title “Composition of Asymmetric RNADuplex as MicroRNA Mimetic or Inhibitor” the entire content of which isincorporated herein by reference).

The novel structural design of aiRNA is not only functionally potent ineffecting gene regulation, but also offers several advantages over thecurrent state-of-art, RNAi regulators (mainly antisense, siRNA). Amongthe advantages, aiRNA can have RNA duplex structure of much shorterlength than the current siRNA constructs, which should reduce the costof synthesis and abrogate or reduce length-dependent triggering ofnonspecific interferon-like immune responses from host cells. Theshorter length of the passenger strand in aiRNA should also eliminate orreduce the passenger strand's unintended incorporation in RISC, and inturn, reduce off-target effects observed in miRNA-mediated genesilencing. AiRNA can be used in all areas that current miRNA-basedtechnologies are being applied or contemplated to be applied, includingbiology research, R&D in biotechnology and pharmaceutical industries,and miRNA-based diagnostics and therapies.

2. the aiRNA Structural Scaffold

Elbashir, et al, tested several asymmetrical duplex RNA molecules aswell as symmetrical siRNA molecules (Elbashir et al., 2001c). Theasymmetrical duplex RNA molecules are listed in table 1 together withthe corresponding siRNA molecules.

TABLE 1 B 5′-CGUACGCGGAAUACUUCG (SEQ ID NO: 60) UGCAUGCGCCUUAUGAAGCUU-5′(SEQ ID NO: 61) 5′-CGUACGCGGAAUACUUCGA (SEQ ID NO: 64)UGCAUGCGCCUUAUGAAGCUU-5′ (SEQ ID NO: 61)5′-CGUACGCGGAAUACUUCGAA (SEQ ID NO: 65) UGCAUGCGCCUUAUGAAGCUU-5′(SEQ ID NO: 61) 5′-CGUACGCGGAAUACUUCGAAA (SEQ ID NO: 66)UGCAUGCGCCUUAUGAAGCUU-5′ (SEQ ID NO: 61)5′-CGUACGCGGAAUACUUCGAAAU (SEQ ID NO: 67) UGCAUGCGCCUUAUGAAGCUU-5′(SEQ ID NO: 61) 5′-CGUACGCGGAAUACUUCGAAAUG (SEQ ID NO: 68)UGCAUGCGCCUUAUGAAGCUU-5′ (SEQ ID NO: 61)5′-CGUACGCGGAAUACUUCGAAAUGU (SEQ ID NO: 69) UGCAUGCGCCUUAUGAAGCUU-5′(SEQ ID NO: 61) 5′-CGUACGCGGAAUACUUCGAAAUGUC (SEQ ID NO: 70)UGCAUGCGCCUUAUGAAGCUU-5′ (SEQ ID NO: 61) C5′-CGUACGCGGAAUACUUCG (SEQ ID NO: 60) GUGCAUGCGCCUUAUGAAGCU-5′(SEQ ID NO: 62) 5′-CGUACGCGGAAUACUUCGA (SEQ ID NO: 64)GUGCAUGCGCCUUAUGAAGCU-5′ (SEQ ID NO: 62)5′-CGUACGCGGAAUACUUCGAA (SEQ ID NO: 65) GUGCAUGCGCCUUAUGAAGCU-5′(SEQ ID NO: 62) 5′-CGUACGCGGAAUACUUCGAAA (SEQ ID NO: 66)GUGCAUGCGCCUUAUGAAGCU-5′ (SEQ ID NO: 62)5′-CGUACGCGGAAUACUUCGAAAU (SEQ ID NO: 67) GUGCAUGCGCCUUAUGAAGCU-5′SEQ ID NO: 62) 5′-CGUACGCGGAAUACUUCGAAAUG (SEQ ID NO: 68)GUGCAUGCGCCUUAUGAAGCU-5′ (SEQ ID NO: 62)5′-CGUACGCGGAAUACUUCGAAAUGU (SEQ ID NO: 69) GUGCAUGCGCCUUAUGAAGCU-5′(SEQ ID NO: 62) 5′-CGUACGCGGAAUACUUCGAAAUGUC (SEQ ID NO: 70)GUGCAUGCGCCUUAUGAAGCU-5′ (SEQ ID NO: 62) D5′-CGUACGCGGAAUACUUCG (SEQ ID NO: 60) AGUGCAUGCGCCUUAUGAAGC-5′(SEQ ID NO: 63) 5′-CGUACGCGGAAUACUUCGA (SEQ ID NO: 64)AGUGCAUGCGCCUUAUGAAGC-5′ (SEQ ID NO: 63)5′-CGUACGCGGAAUACUUCGAA (SEQ ID NO: 65) AGUGCAUGCGCCUUAUGAAGC-5′(SEQ ID NO: 63) 5′-CGUACGCGGAAUACUUCGAAA (SEQ ID NO: 66)AGUGCAUGCGCCUUAUGAAGC-5′ (SEQ ID NO: 63)5′-CGUACGCGGAAUACUUCGAAAU (SEQ ID NO: 67) AGUGCAUGCGCCUUAUGAAGC-5′(SEQ ID NO: 63) 5′-CGUACGCGGAAUACUUCGAAAUG (SEQ ID NO: 68)AGUGCAUGCGCCUUAUGAAGC-5′ (SEQ ID NO: 63)5′-CGUACGCGGAAUACUUCGAAAUGU (SEQ ID NO: 69) AGUGCAUGCGCCUUAUGAAGC-5′(SEQ ID NO: 63) 5′-CGUACGCGGAAUACUUCGAAAUGUC (SEQ ID NO: 70)AGUGCAUGCGCCUUAUGAAGC-5′ (SEQ ID NO: 63)

In comparison with corresponding symmetrical siRNA molecules, however,the asymmetrical duplex RNA molecules are incapable of effectingselective gene silencing (ibid).

The present invention is pertinent to asymmetrical duplex RNA moleculesthat are capable of effecting sequence-specific gene silencing. In anembodiment, a RNA molecule of the present invention comprises a firststrand and a second strand, wherein the second strand is substantiallycomplementary to the first strand, and forms a double-stranded regionwith the first strand, wherein the first strand is longer than thesecond strand; excluding the asymmetrical duplex RNA molecules disclosedin Elbashir (Elbashir et al., 2001c), specifically the asymmetricalduplex RNA molecules listed in Table 1. The RNA molecule comprises adouble-stranded region, and two ends independently selected from thegroup consisting of a 5′-overhang, a 3′-overhang, and a blunt end. TheRNA strand can have unmatched or imperfectly matched nucleotides.

In an embodiment, the first strand is at least 1 nt longer than thesecond strand. In a further embodiment, the first strand is at least 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ntlonger than the second strand. In another embodiment, the first strandis 20-100 nt longer than the second strand. In a further embodiment, thefirst strand is 2-12 nt longer than the second strand. In an evenfurther embodiment, the first strand is 3-10 nt longer than the secondstrand.

In an embodiment, the double-stranded region has a length of 3-98 bp. Ina further embodiment, the double-stranded region has a length of 5-28bp. In an even further embodiment, the double-stranded region has alength of 10-19 bp. The length of the double-stranded region can be 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, or 30 bp.

In an embodiment, the double-stranded region of the RNA molecule doesnot contain any mismatch or bulge. In another embodiment, thedouble-stranded region of the RNA molecule contains mismatch and/orbulge.

In an embodiment, when the first strand is

(SEQ ID NO: 1) 5′-UUCGAAGUAUUCCGCGUACGU (SEQ ID NO: 2)5′-UCGAAGUAUUCCGCGUACGUG or (SEQ ID NO: 3) 5′-CGAAGUAUUCCGCGUACGUGA,

the second strand has a length of at most 17 nucleotides, or contains atleast one mismatch with the first strand, or contains at least onemodification.

In an alternative embodiment, the first strand is not

(SEQ ID NO: 1) 5′-UUCGAAGUAUUCCGCGUACGU (SEQ ID NO: 2)5′-UCGAAGUAUUCCGCGUACGUG or (SEQ ID NO: 3) 5′-CGAAGUAUUCCGCGUACGUGA.

In an embodiment, the first strand comprises a sequence beingsubstantially complimentary to a target triRNA sequence. In anotherembodiment, the second strand comprises a sequence being substantiallycomplimentary to a target mRNA sequence.

The present invention is pertinent to asymmetrical double stranded RNAmolecules that are capable of effecting gene silencing. In anembodiment, an RNA molecule of the present invention comprises a firststrand and a second strand, wherein the second strand is substantiallycomplementary, or partially complementary to the first strand, and thefirst strand and the second strand form at least one double-strandedregion, wherein the first strand is longer than the second strand(length asymmetry). The RNA molecule of the present invention has atleast one double-stranded region, and two ends independently selectedfrom the group consisting of a 5′-overhang, a 3′-overhang, and a bluntend (e.g., see FIGS. 1A, 2A-2D).

In the field of making small RNA regulators where changes, additions anddeletions of a single nucleotide can critically affect the functionalityof the molecule (Elbashir et al., 2001c), the aiRNA scaffold provides astructural platform distinct from the classic siRNA structure of 21-ntdouble-strand RNA which is symmetric in each strand and their respective3′ overhangs. Further, the aiRNA of the present invention provides amuch-needed new approach in designing a new class of small moleculeregulators that, as shown by data included in the examples below, canovercome obstacles currently encountered in RNAi-based researches anddrug development. For example, data from aiRNAs that structurally mimicsiRNAs show that aiRNAs are more efficacious, potent, rapid-onset,durable, and specific than siRNAs in inducing gene silencing.

Any single-stranded region of the RNA molecule of the invention,including any terminal overhangs and gaps in between two double-strandedregions, can be stabilized against degradation, either through chemicalmodification or secondary structure. The RNA strands can have unmatchedor imperfectly matched nucleotides. Each strand may have one or morenicks (a cut in the nucleic acid backbone, e.g., see FIG. 1B), gaps (afragmented strand with one or more missing nucleotides, e.g., see FIG.1C), and modified nucleotides or nucleotide analogues. Not only can anyor all of the nucleotides in the RNA molecule chemically modified, eachstrand may be conjugated with one or more moieties to enhance itsfunctionality, for example, with moieties such as one or more peptides,antibodies, antibody fragments, aptamers, polymers and so on.

In an embodiment, the first strand is at least 1 nt longer than thesecond strand. In a further embodiment, the first strand is at least 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ntlonger than the second strand. In another embodiment, the first strandis 20-100 nt longer than the second strand. In a further embodiment, thefirst strand is 2-12 nt longer than the second strand. In an evenfurther embodiment, the first strand is 3-10 nt longer than the secondstrand.

In an embodiment, the first strand, or the long strand, has a length of5-100 nt, or preferably 10-30 or 12-30 nt, or more preferably 15-28 nt.In one embodiment, the first strand is 21 nucleotides in length. In anembodiment, the second strand, or the short strand, has a length of 3-30nt, or preferably 3-29 nt or 10-26 nt, or more preferably 12-26 nt. Inan embodiment, the second strand has a length of 15 nucleotides.

In an embodiment, the double-stranded region has a length of 3-98 bp. Ina further embodiment, the double-stranded region has a length of 5-28bp. In an even further embodiment, the double-stranded region has alength of 10-19 bp. The length of the double-stranded region can be 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30 bp.

In an embodiment, the double-stranded region of the RNA molecule doesnot contain any mismatch or bulge, and the two strands are perfectlycomplementary to each other in the double-stranded region. In anotherembodiment, the double-stranded region of the RNA molecule containsmismatch and/or bulge.

In an embodiment, the terminal overhang is 1-10 nucleotides. In afurther embodiment, the terminal overhang is 1-8 nucleotides. In anotherembodiment, the terminal overhang is 3 nt.

2.1. the Duplex RNA Molecule with Both a 5′-Overhang and a 3′-Overhangon the First Strand

Referring to FIG. 1A, in one embodiment of the present invention, thedouble stranded RNA molecule has both a 5′-overhang and a 3′-overhang onthe first strand. The RNA molecule comprises a first strand and a secondstrand; the first strand and the second strand form at least onedouble-stranded region with substantially complementary sequences,wherein the first strand is longer than the second strand. On the firststrand, flanking the double-stranded region, there is an unmatchedoverhang on both the 5′ and 3′ termini.

In an embodiment, the first strand is at least 2 nt longer than thesecond strand. In a further embodiment, the first strand is at least 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ntlonger than the second strand. In another embodiment, the first strandis 20-100 nt longer than the second strand. In a further embodiment, thefirst strand is 2-12 nt longer than the second strand. In an evenfurther embodiment, the first strand is 3-10 nt longer than the secondstrand.

In an embodiment, the first strand has a length of 5-100 nt. In afurther embodiment, the first strand has a length of 5-100 nt, and thesecond strand has a length from 3-30 nucleotides. In an even furtherembodiment, the first strand has a length of 5-100 nt, and the secondstrand has a length from 3-18 nucleotides.

In an embodiment, the first strand has a length from 10-30 nucleotides.In a further embodiment, the first strand has a length from 10-30nucleotides, and the second strand has a length from 3-28 nucleotides.In an even further embodiment, the first strand has a length from 10-30nucleotides, and the second strand has a length from 3-19 nucleotides.In an embodiment, the first strand has a length from 12-26 nucleotides.In a further embodiment, the first strand has a length from 12-26nucleotides, and the second strand has a length from 10-24 nucleotides.In an even further embodiment, the first strand has a length from 12-26nucleotides, and the second strand has a length from 10-19 nucleotides.

In an embodiment, the first strand has a length of 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30 nt. In another embodiment, the second strand has a length of3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, or 28 nt.

In an embodiment, the first strand has a length of 21 nt, and the secondstrand has a length of 15 nt.

In an embodiment, the 3′-overhang has a length of 1-10 nt. In a furtherembodiment, the 3′-overhang has a length of 1-8 nt. In an even furtherembodiment, the 3′-overhang has a length of 2-6 nt. In one embodiment,the 3′-overhang has a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt.

In an embodiment, the 5′-overhang has a length of 1-10 nt. In a furtherembodiment, the 5′-overhang has a length of 1-6 nt. In an even furtherembodiment, the 5′-overhang has a length of 2-4 nt. In one embodiment,the 5′-overhang has a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt.

In an embodiment, the length of the 3′-overhang is equal to that of the5′-overhang. In another embodiment, the 3′-overhang is longer than the5′-overhang. In an alternative embodiment, the 3′-overhang is shorterthan the 5′-overhang.

In an embodiment, the duplex RNA molecule comprises a double-strandedregion of substantially complementary sequences of about 15 nt, a 3-nt3′-overhang, and a 3-nt 5′-overhang. The first strand is 21 nt and thesecond strand is 15 nt. In one feature, the double-stranded region ofvarious embodiments consists of perfectly complementary sequences. In analternative feature, the double strand region includes at least one nick(FIG. 1B), gap (FIG. 1C), or mismatch (bulge or loop).

In an embodiment, the double-stranded region has a length of 3-98 bp. Ina further embodiment, the double-stranded region has a length of 5-28bp. In an even further embodiment, the double-stranded region has alength of 10-19 bp. The length of the double-stranded region can be 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, or 30 bp. There can be more than one double-strandedregion.

In an embodiment, the first strand is the antisense strand, which iscapable of targeting a substantially complementary gene transcript suchas a messenger RNA (mRNA) for gene silencing either by cleavage or bytranslation repression.

The present invention also provides a duplex RNA molecule comprising afirst strand with a length from 18-23 nucleotides and a second strandwith a length from 12-17 nucleotides, wherein the second strand issubstantially complementary to the first strand, and forms adouble-stranded region with the first strand, wherein the first strandhas a 3′-overhang from 1-9 nucleotides, and a 5′-overhang from 1-8nucleotides, wherein said duplex RNA molecule is capable of effectingselective gene silencing in a eukaryotic cell. In an embodiment, thefirst strand comprises a sequence being substantially complementary to atarget mRNA sequence.

In an embodiment, the first strand has a length of 20, 21, or 22nucleotides. In another embodiment, the second strand has a length of14, 15, or 16 nucleotides.

In an embodiment, the first strand has a length of 21 nucleotides, andthe second strand has a length of 15 nucleotides. In a furtherembodiment, the first strand has a 3′-overhang of 1, 2, 3, 4, 5, or 6nucleotides. In an even further embodiment, the first strand has a3′-overhang of 3 nucleotides.

2.2. the Duplex RNA Molecule with a Blunt End and a 5′-Overhang or a3′-Overhang on the First Strand

In one embodiment, the duplex RNA molecule comprises a double-strandedregion, a blunt end, and a 5′-overhang or a 3′-overhang (see, e.g.,FIGS. 2A and 2B). The RNA molecule comprises a first strand and a secondstrand, wherein the first strand and the second strand form adouble-stranded region, wherein the first strand is longer than thesecond strand.

In an embodiment, the double-stranded region has a length of 3-98 bp. Ina further embodiment, the double-stranded region has a length of 5-28bp. In an even further embodiment, the double-stranded region has alength of 10-18 bp. The length of the double-stranded region can be 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, or 30 bp. The double-stranded region can havefeatures similar to those described with regard to other embodiments andare not necessarily repeated here. For example, the double-strandedregion can consist of perfectly complementary sequences or include atleast one nick, gap, or mismatch (bulge or loop).

In an embodiment, the first strand is at least 1 nt longer than thesecond strand. In a further embodiment, the first strand is at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ntlonger than the second strand. In another embodiment, the first strandis 20-100 nt longer than the second strand. In a further embodiment, thefirst strand is 2-12 nt longer than the second strand. In an evenfurther embodiment, the first strand is 4-10 nt longer than the secondstrand.

In an embodiment, the first strand has a length of 5-100 nt. In afurther embodiment, the first strand has a length of 5-100 nt, and thesecond strand has a length from 3-30 nucleotides. In an even furtherembodiment, the first strand has a length of 10-30 nt, and the secondstrand has a length from 3-19 nucleotides. In another embodiment, thefirst strand has a length from 12-26 nucleotides, and the second strandhas a length from 10-19 nucleotides.

In an embodiment, the duplex RNA molecule comprises a double-strandedregion, a blunt end, and a 3′-overhang (see, e.g., FIG. 2B).

In an embodiment, the 3′-overhang has a length of 1-10 nt. In a furtherembodiment, the 3′-overhang has a length of 1-8 nt. In an even furtherembodiment, the 3′-overhang has a length of 2-6 nt. In one embodiment,the 3′-overhang has a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt.

In an alternative embodiment, the duplex RNA molecule comprises adouble-stranded region, a blunt end, and a 5′-overhang (see, e.g., FIG.2A).

In an embodiment, the 5′-overhang has a length of 1-10 nt. In a furtherembodiment, the 5′-overhang has a length of 1-6 nt. In an even furtherembodiment, the 5′-overhang has a length of 2-4 nt. In one embodiment,the 5′-overhang has a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt.

2.3. the Duplex RNA Molecule with Two 5′-Overhangs or Two 3′-Overhangs

In one embodiment, the duplex RNA molecule comprises a double-strandedregion, and two 3′-overhangs or two 5′-overhangs (see, e.g., FIGS. 2Cand 2D). The RNA molecule comprises a first strand and a second strand,wherein the first strand and the second strand form a double-strandedregion, wherein the first strand is longer than the second strand.

In an embodiment, the double-stranded region has a length of 3-98 bp. Ina further embodiment, the double-stranded region has a length of 5-28bp. In an even further embodiment, the double-stranded region has alength of 10-18 bp. The length of the double-stranded region can be 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, or 30 bp.

In an embodiment, the first strand is at least 1 nt longer than thesecond strand. In a further embodiment, the first strand is at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ntlonger than the second strand. In another embodiment, the first strandis 20-100 nt longer than the second strand. In a further embodiment, thefirst strand is 2-12 nt longer than the second strand. In an evenfurther embodiment, the first strand is 4-10 nt longer than the secondstrand.

In an embodiment, the first strand has a length of 5-100 nt. In afurther embodiment, the first strand has a length of 5-100 nt, and thesecond strand has a length from 3-30 nucleotides. In an even furtherembodiment, the first strand has a length of 10-30 nt, and the secondstrand has a length from 3-18 nucleotides. In another embodiment, thefirst strand has a length from 12-26 nucleotides, and the second strandhas a length from 10-16 nucleotides.

In an alternative embodiment, the duplex RNA molecule comprises adouble-stranded region, and two 3′-overhangs (see, e.g., FIG. 2C). Thedouble-stranded region shares similar features as described with regardto other embodiments.

In an embodiment, the 3′-overhang has a length of 1-10 nt. In a furtherembodiment, the 3′-overhang has a length of 1-6 nt. In an even furtherembodiment, the 3′-overhang has a length of 2-4 nt. In one embodiment,the 3′-overhang has a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt.

In an embodiment, the duplex RNA molecule comprises a double-strandedregion, and two 5′-overhangs (see, e.g., FIG. 2D).

In an embodiment, the 5′-overhang has a length of 1-10 nt. In a furtherembodiment, the 5′-overhang has a length of 1-6 nt. In an even furtherembodiment, the 5′-overhang has a length of 2-4 nt. In one embodiment,the 3′-overhang has a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt.

-   3. The design of aiRNAs

siRNAs and miRNAs are widely used as research tools, and developed asdrug candidates. (see, e.g., Dykxhoorn, Novina & Sharp. Nat. Rev. Mol.cell Biol. 4:457-467 (2003); Kim & Rossi, Nature Rev. Genet. 8:173-184(2007); de Fougerolles, et al. Nature Rev. Drug Discov. 6:443-453(2007); Czech, NEJM 354:1194-1195 (2006); and Mack, Nature Biotech.25:631-638 (2007)). The duplex RNA molecules of the present invention,i.e., aiRNAs, can be derived from siRNAs and miRNAs known in the field.

The present invention provides a method of converting an siRNA or amiRNA into an aiRNA. The conversion results in a new duplex RNA moleculethat has at least one property improved in comparison to the originalmolecule. The property can be size, efficacy, potency, the speed ofonset, durability, synthesis cost, off-target effects, interferonresponse, or delivery.

In an embodiment, the original molecule is a duplex RNA molecule, suchas an siRNA. The duplex RNA molecule comprises an antisense strand(e.g., a guide strand) and a sense strand (e.g. a passenger strand) thatform at least one double-stranded region. The method comprises changingthe length of one or both strands so that the antisense strand is longerthan the sense strand. In an embodiment, sense passenger strand isshortened. In another embodiment, the antisense strand is elongated. Inan even further embodiment, the sense strand is shortened and theantisense strand is elongated. The antisense and sense RNA strands,intact or with changed size, can be synthesized, and then combined underconditions, wherein an aiRNA molecule is formed.

In a further embodiment, the method comprises changing the length of theantisene and/or sense strand so that the duplex RNA molecule is formedhaving at least one of a 3′-overhang of 1-6 nucleotides and a5′-overhang of 1-6 nucleotides.

Alternatively, the duplex RNA molecules of the present invention can bedesigned de novo. A duplex RNA molecule of the present invention can bedesigned taking advantage of the design methods for siRNAs and miRNAs,such as the method of gene walk.

An RNA molecule of the present invention can be designed withbioinformatics approaches, and then tested in vitro and in vivo todetermine its modulating efficacy against the target gene and theexistence of any off-target effects. Based on these studies, thesequences of the RNA molecules can then be selected and modified toimprove modulating efficacy against the target gene, and to minimizeoff-target effects. (see e.g., Patzel, Drug Discovery Today 12:139-148(2007)).

3.1. Unmatched or Mismatched Region in the Duplex RNA Molecule

The two single strands of the aiRNA duplex can have at least oneunmatched or imperfectly matched region containing, e.g., one or moremismatches. In one embodiment, the unmatched or imperfectly matchedregion is at least one end region of the RNA molecule, including an endregion with a blunt end, an end region with a 3′-recess or a 5′overhang, and an end region with a 5′ recess or a 3′ overhang. As usedherein, the end region is a region of the RNA molecule including one endand the neighboring area.

In an embodiment, the unmatched or imperfectly matched region is in adouble-stranded region of the aiRNA molecule. In a further embodiment,the asymmetric RNA duplex has an unmatched bulge or loop structure.

3.2. Sequence Motifs in the Duplex RNA Molecule

In the design of an aiRNA molecule of the invention, the overall GCcontent may vary. In an embodiment, the GC content of thedouble-stranded region is 20-70%. In a further embodiment, the GCcontent of the double-stranded region is less than 50%, or preferably30-50%, to make it easier for strand separation as the G-C pairing isstronger than the A-U pairing.

The nucleotide sequence at a terminal overhang, in some embodiments,e.g., the 5′ terminal, can be designed independently from any templatesequence (e.g., a target mRNA sequence), i.e., does not have to besubstantially complementary to a target tnRNA (in the case of an siRNAor miRNA mimetic) or a target miRNA (in the case of miRNA inhibitor). Inone embodiment, the overhang, e.g., at the 5′ or the 3′, of the longeror antisense strand, is an “AA”, “UU” or “dTdT” motif, which haveexhibited increased efficacy in comparison to some other motifs. In anembodiment, the 5′ overhang of the longer or antisense strand has an“AA” motif. In another embodiment, the 3′ overhang of the longer orantisense strand has a “UU” motif.

3.3. Nucleotide Substitution

One or more of the nucleotides in the RNA molecule of the invention canbe substituted with deoxynucleotides or modified nucleotides ornucleotide analogues. The substitution can take place anywhere in theRNA molecule, e.g., one or both of the overhang regions, and/or adouble-stranded region. In some cases, the substitution enhances aphysical property of the RNA molecule such as strand affinity,solubility and resistance to RNase degradation or enhanced stabilityotherwise.

In one embodiment, the modified nucleotide or analogue is a sugar-,backbone-, and/or base-modified ribonucleotide. The backbone-modifiedribonucleotide may have a modification in a phosphodiester linkage withanother ribonucleotide. In an embodiment, the phosphodiester linkage inthe RNA molecule is modified to include at least a nitrogen and/orsulphur heteroatom. In an embodiment, the modified nucleotide oranalogue is an unusual base or a modified base. In an embodiment, themodified nucleotide or analogue is inosine, or a tritylated base.

In a further embodiment, the nucleotide analogue is a sugar-modifiedribonucleotide in which the 2′-OH group is replaced by a group selectedfrom the group consisting of H, OR, R, halo, SH, SR, NH₂, NHR, NR₂, andCN, wherein each R is independently selected from the group consistingof C1-C6 alkyl, alkenyl and alkynyl, and halo is selected from the groupconsisting of F, Cl, Br and I.

In one embodiment, the nucleotide analogue is a backbone-modifiedribonucleotide containing a phosphothioate group.

4. The utilities

The present invention also provides a method of modulating geneexpression in a cell or an organism (silencing method). The methodcomprises the steps of contacting said cell or organism with the duplexRNA molecule under conditions wherein selective gene silencing canoccur, and mediating a selective gene silencing effected by the saidduplex RNA molecule towards a target nucleic acid having a sequenceportion substantially corresponding to the double-stranded RNA.

In an embodiment, the contacting step comprises the step of introducingsaid duplex RNA molecule into a target cell in culture or in an organismin which the selective gene silencing can occur. In a furtherembodiment, the introducing step comprises transfection, lipofection,infection, electroporation, or other delivery technologies.

In an embodiment, the silencing method is used for determining thefunction or utility of a gene in a cell or an organism.

The silencing method can be used for modulating the expression of a genein a cell or an organism. In an embodiment, the gene is associated witha disease, e.g., a human disease or an animal disease, a pathologicalcondition, or an undesirable condition. In a further embodiment, thegene is a gene of a pathogenic microorganism. In an even furtherembodiment, the gene is a viral gene. In another embodiment, the gene isa tumor-associated gene.

In an alternative embodiment, the gene is a gene associated withautoimmune disease, inflammatory diseases, degenerative diseases,infectious diseases, proliferative diseases, metabolic diseases,immune-mediated disorders, allergic diseases, dermatological diseases,malignant diseases, gastrointestinal disorders, respiratory disorders,cardiovascular disorders, renal disorders, rheumatoid disorders,neurological disorders, endocrine disorders, and aging.

4.1. Research Tools

The RNA molecules of the present invention can be used to create gene“knockdown” in animal models as opposed to genetically engineeredknockout models to discover gene functions. The methods can also be usedto silence genes in vitro. For example, aiRNA can be transfected tocells. AiRNA can be used to I as a research tool in drug target/pathwayidentification and validation, and other biomedical research in drugresearch and development.

4.2 Therapeutic Uses

The RNA molecules of the present invention can be used for the treatmentand or prevention of various diseases or undesirable conditions,including the diseases summarized (Czech, 2006; de Fougerolles et al.,2007; Dykxhoorn et al., 2003; Kim and Rossi, 2007; Mack, 2007).

In an embodiment, the present invention can be used as a cancer therapyor to prevent cancer. The RNA molecules of the present invention can beused to silence or knock down genes involved with cell proliferation orother cancer phenotypes. Examples of these genes are k-Ras, β-catenin,Nbs1, EF2, Stat3, PTEN, p70S6K, mTOR, Rsk1, PCNA, Parp1, Survivin, NQO1,and p21. Specifically, k-Ras and β-catenin are therapeutic genes ofcolon cancer. These oncogenes are active and relevant in the majority ofclinical cases.

These RNA molecules can also be used to silence or knockdown non-cancergene targets. The RNA molecules of the invention can also be used totreat or prevent ocular diseases, (e.g., age-related maculardegeneration (AMD) and diabetic retinopathy (DR)); infectious diseases(e.g. HIV/AIDS, hepatitis B virus (HBV), hepatitis C virus (HCV), humanpapillomavirus (HPV), herpes simplex virus (HSV), RCV, cytomegalovirus(CMV), dengue fever, west Nile virus); respiratory diseases (e.g.,respiratory syncytial virus (RSV), asthma, cystic fibrosis);neurological diseases (e.g., Huntingdon's disease (HD), amyotrophiclateral sclerosis (ALS), spinal cord injury, Parkinson's disease,Alzheimer's disease, pain); cardiovascular diseases; metabolic disorders(e.g., diabetes); genetic disorders; and inflammatory conditions (e.g.,inflammatory bowel disease (IBD), arthritis, rheumatoid disease,autoimmune disorders), dermatological diseases.

Various genes can be silenced using the asymmetrical duplex RNA moleculeof the present invention. In an embodiment, the first strand comprises asequence being substantially complimentary to the target mRNA sequenceof a gene selected from the group consisting of a developmental gene, anoncogene, a tumor suppresser gene, and an enzyme gene, and a gene for anadhesion molecule, a cyclin kinase inhibitor, a Wnt family member, a Paxfamily member, a Winged helix family member, a Hox family member, acytokine/lymphokine or its receptor, a growth/differentiation factor orits receptor, a neurotransmitter or its receptor, ABLI, BCL1, BCL2,BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2, ETS1, ETS1, ETV6, FGR, FOS,FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCL1, MYCN,NRAS, PIM1, PML, RET, SRC, TAL1, TCL3 and YES) (e.g., APC, BRCA1, BRCA2,MADH4, MCC, NF1, NF2, RB1, TP53, WT1, an ACP desaturase or hydroxylase,an ADP-glucose pyrophorylase, an ATPase, an alcohol dehydrogenase, anamylase, an amyloglucosidase, a catalase, a cellulase, a cyclooxygenase,a decarboxylase, a dextrinase, a DNA or RNA polymerase, a galactosidase,a glucanase, a glucose oxidase, a GTPase, a helicase, a hemicellulase,an integrase, an invertase, an isomerase, a kinase, a lactase, a lipase,a lipoxygenase, a lysozyme, a pectinesterase, a peroxidase, aphosphatase, a phospholipase, a phosphorylase, a polygalacturonase, aproteinase or peptideases, a pullanase, a recombinase, a reversetranscriptase, a topoisomerase, a xylanase, k-RAS, β-Catenin, Rsk1,PCNA, p70S6K, Survivin, mTOR, PTEN, Parp1, or p21.

The present invention provides a method to treat a disease orundesirable condition. The method comprises using the asymmetricalduplex RNA molecule to effect gene silencing of a gene associated withthe disease or undesirable condition.

4.3. Converting the RNA Molecules (aiRNA) into Drugs

4.3.1. Modifications of the RNA Molecules

Naked RNA molecules are relatively unstable and can be degraded in vivorelatively quickly. Chemical modifications can be introduced to the RNAmolecules of the present invention to improve their half-life and tofurther reduce the risk of non-specific effects of gene targeting,without reducing their biological activities.

The modifications of RNA molecules have been investigated to improve thestability of various RNA molecules, including antisense RNA, ribozyme,aptamer, and RNAi (Chiu and Rana, 2003; Czauderna et al., 2003; deFougerolles et al., 2007; Kim and Rossi, 2007; Mack, 2007; Zhang et al.,2006; and Schmidt, Nature Biotech. 25:273-275 (2007))

Any stabilizing modification known to a skill in the art can be used toimprove the stability of the RNA molecules of the present invention.Within the RNA molecules of the present invention, chemicalmodifications can be introduced to the phosphate backbone (e.g.,phosphorothioate linkages), the ribose (e.g., locked nucleic acids,2′-deoxy-2′-fluorouridine, 2′-O-methyl), and/or the base (e.g.,2′-fluoropyrimidines). Several examples of such chemical modificationsare summarized in the following.

Chemical modifications at the 2′ position of the ribose, such as2′-O-methylpurines and 2′-fluoropyrimidines, which increase resistanceto endonuclease activity in serum, can be adopted to the stabilize theRNA molecules of the present invention. The position for theintroduction of the modification should be carefully selected to avoidsignificantly reducing the silencing potency of the RNA molecule. Forexample, the modifications on 5′ end of the guide strand can reduce thesilencing activity. On the other hand, 2′-O-methyl modifications can bestaggered between the two RNA strands at the double-stranded region toimprove the stability while reserving the gene silencing potency. The2′-O-methyl modifications can also eliminate or reduce the interferoninduction.

Another stabilizing modification is phosphorothioate (P═S) linkage. Theintroduction of phosphorothioate (P═S) linkage into the RNA molecules,e.g., at the 3′-overhang, can provide protection against exonuclease.

The introduction of deoxyribonucleotides into the RNA molecules can alsoreduce the manufacture cost, and increase stability.

In an embodiment, the 3′-overhang, 5′-overhang, or both are stabilizedagainst degradation.

In an embodiment, the RNA molecule contains at least one modifiednucleotide or its analogue. In a further embodiment, the modifiedribonucleotide is modified at its sugar, backbone, base, or anycombination of the three.

In an embodiment, the nucleotide analogue is a sugar-modifiedribonucleotide. In a further embodiment, the 2′-OH group of thenucleotide analogue is replaced by a group selected from H, OR, R, halo,SH, SR, NH₂, NHR, NR₂, or CN, wherein each R is independently C1-C6alkyl, alkenyl or alkynyl, and halo is F, Cl, Br or I.

In an alternative embodiment, the nucleotide analogue is abackbone-modified ribonucleotide containing a phosphothioate group.

In an embodiment, the duplex RNA molecule contains at least onedeoxynucleotide. In a further embodiment, the first strand comprises 1-6deoxynucleotides. In an even further embodiment, the first strandcomprises 1-3 deoxynucleotides. In another embodiment, the 3′-overhangcomprises 1-3 deoxynucleotides. In a further embodiment, the 5′-overhangcomprises 1-3 deoxynucleotides. In an alternative embodiment, the secondstrand comprises 1-5 deoxynucleotides.

In an embodiment, the duplex RNA molecule comprises a 3′-overhang or5′-overhang that contains at least one deoxynucleotide. In anotherembodiment, the RNA the 3′-overhang and/or 5′-overhang consists ofdeoxynucleotides.

In an embodiment, the duplex RNA molecule is conjugated to an entity. Ina further embodiment, the entity is selected from the group consistingof peptide, antibody, polymer, lipid, oligonucleotide, and aptamer.

In another embodiment, the first strand and the second strand are joinedby a chemical linker.

4.4. In Vivo Delivery of the RNA Molecules

One major obstacle for the therapeutic use of RNAi is the delivery ofsiRNA to the target cell (Zamore and Aronin, 2003). Various approacheshave been developed for the delivery of RNA molecules, especially siRNAmolecules (de Fougerolles et al., 2007; Dykxhoom et al., 2003; Kim andRossi, 2007). Any delivery approach known to a skill of the art can beused for the delivery of the RNA molecules of the present invention.

Major issues in delivery include instability in senim, non-specificdistribution, tissue barriers, and non-specific interferon response (Lu& Woodle, Methods in Mol Biology 437: 93-107 (2008)). Compared to theirsiRNA and miRNA counterparts, aiRNA molecules possess several advantagesthat should make a wider ranger of methods available for deliverypurpose. First, aiRNAs can be designed to be smaller than their siRNAand miRNA counterparts, therefore, reducing or eliminating anyinterferon responses. Second, aiRNAs are more potent, faster-onsetting,more efficacious and lasts longer, therefore, less amount/dosage ofaiRNAs is required to achieve a therapeutic goal. Third, aiRNA aredouble stranded and more stable than single-stranded antisense oligosand miRNAs, and they can be further modified chemically to enhancestability. Therefore, the RNA molecules of the invention can bedelivered into a subject via a variety of systemic or local deliveryroutes. In some embodiments, molecules of the invention are deliveredthrough systemic delivery routes include intra-venous (I.V.) andintra-peritoneal (ip). In other embodiments, molecules of the inventionare delivered through local delivery routes, e.g., intra-nasal,intra-vitreous, intra-tracheal, intra-cerebral, intra-muscle,intra-articular, and intra-tumor.

Examples of the delivery technologies include direct injection of nakedRNA molecules, conjugation of the RNA molecules to a natural ligand suchas cholesterol, or an aptamer, liposome-formulated delivery, andnon-covalently binding to antibody-protamine fusion proteins. Othercarrier choices include positive charged carriers (e.g., cationic lipidsand polymers) and various protein carriers. In one embodiment, thedelivery of the molecules of the invention uses a ligand-targeteddelivery system based on the cationic liposome complex or polymercomplex systems (Woodle, et al. J Control Release 74: 309-311; Song, etal. Nat Biotechnol. 23(6): 709-717 (2005); Morrissey et al. NatBiotechnol. 23(8): 1002-1007 (2005)).

In one embodiment, molecules of the invention is formulated with acollagen carrier, e.g., atelocollagen, for in vivo delivery.Atelocollagen has been reported to protect siRNA from being digested byRNase and to enable sustained release (Minakuchi, et al. Nucleic AcidsRes. 32: e109 (2004); Takei et al. Cancer Res. 64: 3365-3370 (2004)). Inanother embodiment, molecules of the invention are formulated withnanoparticles or form a nanoemulsion, e.g., RGD peptide ligand targetednanoparticles. It has been shown that different siRNA oligos can becombined in the same RGD ligand targeted nanoparticle to target severalgenes at the same time (Woodle et al. Materials Today 8 (suppl 1): 34-41(2005)).

Viral vectors can also be used for the delivery of the RNA molecules ofthe present invention. In an embodiment, lentiviral vectors are used todeliver the RNA molecule transgenes that integrate into the genome forstable expression. In another embodiment, adenoviral andadeno-associated virus (AAV) are used to deliver the RNA moleculetransgenes that do not integrate into the genome and have episomalexpression.

Moreover, bacteria can be used for the delivery of the RNA molecules ofthe present invention (Xiang et al., 2006).

5. the Pharmaceutical Compositions and Formulations

The present invention further provided a pharmaceutical composition. Thepharmaceutical comprises as an active agent at least one asymmetricalduplex RNA molecule and one or more carriers selected from the groupconsisting of a pharmaceutical carrier, a positive-charge carrier, aliposome, a protein carrier, a polymer, a nanoparticle, a nanoemulsion,a lipid, and a lipoid. In an embodiment, the composition is fordiagnostic applications, or for therapeutic applications.

The pharmaceutical compositions and formulations of the presentinvention can be the same or similar to the pharmaceutical compositionsand formulations developed for siRNA, miRNA, and antisense RNA (deFougerolles et al., 2007; Kim and Rossi, 2007), except for the RNAingredient. The siRNA, miRNA, and antisense RNA in the pharmaceuticalcompositions and formulations can be replaced by the duplex RNAmolecules of the present information. The pharmaceutical compositionsand formulations can also be further modified to accommodate the duplexRNA molecules of the present information.

A “pharmaceutically acceptable salt” or “salt” of the disclosed duplexRNA molecule is a product of the disclosed duplex RNA molecule thatcontains an ionic bond, and is typically produced by reacting thedisclosed duplex RNA molecule with either an acid or a base, suitablefor administering to a subject. Pharmaceutically acceptable salt caninclude, but is not limited to, acid addition salts includinghydrochlorides, hydrobromides, phosphates, sulphates, hydrogensulphates, alkylsulphonates, arylsulphonates, acetates, benzoates,citrates, maleates, fumarates, succinates, lactates, and tartrates;alkali metal cations such as Na, K, Li, alkali earth metal salts such asMg or Ca, or organic amine salts.

A “pharmaceutical composition” is a formulation containing the disclosedduplex RNA molecules in a form suitable for administration to a subject.In one embodiment, the pharmaceutical composition is in bulk or in unitdosage form. The unit dosage form is any of a variety of forms,including, for example, a capsule, an IV bag, a tablet, a single pump onan aerosol inhaler, or a vial. The quantity of active ingredient (e.g.,a formulation of the disclosed duplex RNA molecule or salts thereof) ina unit dose of composition is an effective amount and is variedaccording to the particular treatment involved. One skilled in the artwill appreciate that it is sometimes necessary to make routinevariations to the dosage depending on the age and condition of thepatient. The dosage will also depend on the route of administration. Avariety of routes are contemplated, including oral, pulmonary, rectal,parenteral, transdermal, subcutaneous, intravenous, intramuscular,intraperitoneal, intranasal, and the like. Dosage forms for the topicalor transdermal administration of a duplex RNA molecule of this inventioninclude powders, sprays, ointments, pastes, creams, lotions, gels,solutions, patches and inhalants. In one embodiment, the active duplexRNA molecule is mixed under sterile conditions with a pharmaceuticallyacceptable carrier, and with any preservatives, buffers, or propellantsthat are required.

The present invention provides a method of treatment comprisingadministering an effective amount of the pharmaceutical composition to asubject in need. In an embodiment, the pharmaceutical composition isadministered via a route selected from the group consisting of iv, sc,topical, po, and ip. In another embodiment, the effective amount is 1 ngto 1 g per day, 100 ng to 1 g per day, or 1 μg to 1 mg per day.

The present invention also provides pharmaceutical formulationscomprising a duplex RNA molecule of the present invention in combinationwith at least one pharmaceutically acceptable excipient or carrier. Asused herein, “pharmaceutically acceptable excipient” or“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. Suitable carriers aredescribed in “Remington: The Science and Practice of Pharmacy, TwentiethEdition,” Lippincott Williams & Wilkins, Philadelphia, Pa., which isincorporated herein by reference. Examples of such carriers or diluentsinclude, but are not limited to, water, saline, Ringer's solutions,dextrose solution, and 5% human serum albumin. Liposomes and non-aqueousvehicles such as fixed oils may also be used. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active duplex RNA molecule, use thereof in the compositions iscontemplated. Supplementary active duplex RNA molecules can also beincorporated into the compositions.

Methods for formulation are disclosed in PCT International ApplicationPCT/US02/24262 (WO03/011224), U.S. Patent Application Publication No.2003/0091639 and U.S. Patent Application Publication No. 2004/0071775,each of which is incorporated by reference herein.

A duplex RNA molecule of the present invention is administered in asuitable dosage form prepared by combining a therapeutically effectiveamount (e.g., an efficacious level sufficient to achieve the desiredtherapeutic effect through inhibition of tumor growth, killing of tumorcells, treatment or prevention of cell proliferative disorders, etc.) ofa duplex RNA molecule of the present invention (as an active ingredient)with standard pharmaceutical carriers or diluents according toconventional procedures (i.e., by producing a pharmaceutical compositionof the invention). These procedures may involve mixing, granulating, andcompressing or dissolving the ingredients as appropriate to attain thedesired preparation. In another embodiment, a therapeutically effectiveamount of a duplex RNA molecule of the present invention is administeredin a suitable dosage form without standard pharmaceutical carriers ordiluents.

Pharmaceutically acceptable carriers include solid carriers such aslactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia,magnesium stearate, stearic acid and the like. Exemplary liquid carriersinclude syrup, peanut oil, olive oil, water and the like. Similarly, thecarrier or diluent may include time-delay material known in the art,such as glyceryl monostearate or glyceryl distearate, alone or with awax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate orthe like. Other fillers, excipients, flavorants, and other additivessuch as are known in the art may also be included in a pharmaceuticalcomposition according to this invention.

The pharmaceutical compositions containing active duplex RNA moleculesof the present invention may be manufactured in a manner that isgenerally known, e.g., by means of conventional mixing, dissolving,granulating, dragee-making, levigating, emulsifying, encapsulating,entrapping, or lyophilizing processes. Pharmaceutical compositions maybe formulated in a conventional manner using one or more physiologicallyacceptable carriers comprising excipients and/or auxiliaries whichfacilitate processing of the active duplex RNA molecules intopreparations that can be used pharmaceutically. Of course, theappropriate formulation is dependent upon the route of administrationchosen.

A duplex RNA molecule or pharmaceutical composition of the invention canbe administered to a subject in many of the well-known methods currentlyused for chemotherapeutic treatment. For example, for treatment ofcancers, a duplex RNA molecule of the invention may be injected directlyinto tumors, injected into the blood stream or body cavities or takenorally or applied through the skin with patches. For treatment ofpsoriatic conditions, systemic administration (e.g., oraladministration), or topical administration to affected areas of theskin, are preferred routes of administration. The dose chosen should besufficient to constitute effective treatment but not as high as to causeunacceptable side effects. The state of the disease condition (e.g.,cancer, psoriasis, and the like) and the health of the patient should beclosely monitored during and for a reasonable period after treatment.

EXAMPLES

Examples are provided below to further illustrate different features ofthe present invention. The examples also illustrate useful methodologyfor practicing the invention. These examples do not limit the claimedinvention.

RNA interference (RNAi) is a catalytic mechanism of gene-specificsilencing in eukaryotic organisms with profound implications for biologyand medicine (Fire et al., 1998), 12. RNAi is mediated by theRNA-induced silencing complex (RISC) (Hammond et al., 2000; Martinez andTuschl, 2004; Rana, 2007) upon incorporating with small interfering RNAs(siRNA) of 19-21 base pairs (bp) with 3′ overhangs, the smallest RNAduplex known to enter RISC and mediate RNAi (Elbashir et al., 2001a;Elbashir et al., 2001b; Elbashir et al., 2001c; Fire et al., 1998;Zamore et al., 2000). As the natural substrate of the RISC enzymecomplex, siRNA can be chemically synthesized or generated throughDicer-catalyzed processing of its various precursors (Donze and Picard,2002; Hammond et al., 2000; Kim et al., 2005; Paddison et al., 2002).While being used widely for gene silencing, siRNA has limited efficiencyin gene silencing with low silencing efficacy for numerous genes inmammalian cells (de Fougerolles et al., 2007; Iorns et al., 2007). Herewe investigate structural scaffold requirements for an efficient RNAimediator in mammalian cells. To our surprise, we found that asymmetricRNA duplexes of 14-15 bp with dual antisense overhangs mediate potentand efficacious gene silencing in mammalian cells. The asymmetricinterfering RNA (aiRNA), structurally characterized by duplex RNA of14-15 by with 3′ and 5′ antisense overhangs, was incorporated into RISCwith higher efficiency than siRNA. The aiRNA caused sequence-specificcleavage of the mRNA and targeted gene silencing in mammalian cells.When the identical sequence of β-catenin mRNA was targeted, the aiRNAwas more efficacious (near 100%), potent (picoM), rapid-onset (less than24 h) and durable (up to 1 week) than siRNA in mediating gene silencingin vitro. These results suggest aiRNA as the smallest RNA duplexscaffold incorporated into RISC and non-siRNA type of RNAi mediatorsthat silence genes with better efficiency than siRNA in mammalian cells.Therefore, aiRNA may have significant potential for broad RNAiapplication.

Methods and Materials

Cell Culture and Reagents

Hela, SW480, DLD1, HT29, and H1299 cells were obtained from ATCC, andcultured in Dulbecco's modified Eagle's medium (DMEM) containing 10%fetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/mlstreptomycin and 2 mM L-glutamine (Invitrogen). Fresh peripheral bloodmononuclear cells (PBMC) were obtained from AllCells LLC and maintainedin RPMI-1640 medium containing 10% FBS and pen/strep (Invitrogen). SmallRNAs described in this study were synthesized by Dharmacon, Qiagen, orIntegrated DNA technologies (Table 2) and annealed following themanufacturer's instructions (FIG. 3a ). siRNAs targeting human Ago2, andDicer (Ambion) were used at 100 nM. Transfections of the RNAs wereperformed using DharmaFECT1 (Dhannacon) at the indicated concentrations.Human Argonaute2 (Ago2) expression vector (OriGene) was transfectedusing Lipofectamine 2000 (Invitrogen). Serum stability was determined byincubation of aiRNA or siRNA duplex with 10% human serum (Sigma) for theindicated amount of time followed by non-denaturing TBE-acrylamide gelelectrophoresis and ethidium bromide staining.

Northern Blot Analysis.

To determine levels of β-catenin, total RNA was extracted with TRIZOL(Invitrogen) from siRNA or aiRNA transfected Hela cells at various timepoints. 20 μg of total cellular RNA was loaded to each lane of adenaturing agarose gel. After electrophoresis, RNA was transferred toHybond-XL Nylon membrane (Amersham Biosciences), UV crosslinked, andbaked at 80° C. for 30 min. Probes detecting β-catenin and actin mRNAwas prepared using Prime-It II Random Primer Labeling Kit (Stratagene)from β-cantenin cDNA fragment (1-568 nt) and actin cDNA fragment (1-500nt). To analyze small RNA RISC loading, siRNA or aiRNA were transfectedinto Hela cells 48 hours after transfection with pCMV-Ago2. Cells werelysed at the indicated timepoints and immunoprecipitated with Ago2antibody. Immunoprecipitates were washed, RNA isolated from the complexby TRIZOL extraction, and loaded on a 15% TBE-Urea PAGE gel (Bio-Rad).Following electrophoreses, RNA was transferred to Hybond-XL Nylonmembrane. mirVana miRNA Probe Kit (Ambion) was used to generate 5′ ³²Plabeled RNA probes. Antisense probe (5′-GUAGCUGAUAUUGAUGGACUU-3′ (SEQ IDNO:71)). Sense probe (5′-UCCAUCAAUAUCAGC-3′ (SEQ ID NO:72))

In Vitro Ago2-RISC Loading.

aiRNA or siRNA sense and anti-sense strands were ³²P end labeled usingT4 kinase (Promega). End labeled RNAs were purified byphenol/chloroform/isoamyl alcohol, precipitated with EtOH, andresuspended in water. Labeled RNAs were then annealed to siRNA or aiRNAanti-sense strands as described. For in vitro lysates, Hela cells weretransfected for 24 hours with human Ago2 expression vector, and S10lysates generated essentially as described (Dignam et al., 1983). 5′sense strand or anti-sense strand labeled duplex aiRNA or siRNA was thenadded to the Ago2-S10 lysate. Following a 5 min incubation at 37° C.,Ago2 was immunoprecipited as described, and Ago2-associated (pellet) andnon-Ago2 associated (supernatant) fractions were separated on a 20%TBE-acrylamide gel and gel exposed to film to detect sense strand-Ago2association. For aiRNA and siRNA competition experiments, up to 100folds cold aiRNA and siRNA were used to compete with ³²P labeled aiRNAor siRNA to load to RISC. Briefly, S10 lysates were generated from Helacells transfected with Ago2 expression vector as described. LabeledaiRNA or siRNA was then added to the S10 lysates followed immediately byaddition of unlabeled aiRNA or siRNA. Reaction was incubated for 5 minat 37° C. and processed as described above.

qRT-PCR.

Cells transfected with the indicated aiRNA or siRNA were harvested atthe indicated time points following transfection. RNA was isolated withTRIZOL, and qRT-PCR performed using TaqMan one-step RT-PCR reagents andprimer probe sets for the indicated mRNA (Applied Biosystems). Data ispresented relative to control transfected cells and each sample isnormalized to actin mRNA levels. For the experiment in FIG. 14d , Stat3constructs were created by cloning Stat3 cDNA (Origene) into eitherpcDNA3.1⁺ or pcDNA3.1⁻ at the HindIII-Xho1 sites. Stat3 forward orreverse expression vectors were then co-transfected into Hela cells withaiStat3 or siStat3 for 24 hours. Cells were then harvested, RNA isolatedby TRIZOL, and qRT-PCR performed using TaqMan one-step RT-PCR reagentsand primer probe sets for Stat3 or actin (Applied Biosystems). RT-PCRwas performed on the same RNA samples using Superscript One-Step RT-PCRkit (Invitrogen) and Stat3 forward (5′-GGATCTAGAATCAGCTACAGCAGC-3′ (SEQID NO:73)) and Stat3 reverse (5% TCCTCTAGAGGGCAATCTCCATTG-3′ (SEQ IDNO:74)) primers and actin forward (5′-CCATGGATGATGATATCGCC-3′ (SEQ IDNO:75)) and actin reverse (5% TAGAAGCATITGCGGIGGAC-3′ (SEQ ID NO:76))primers.

RT-PCR.

Total RNA was prepared using the TRIZOL, and cDNA was synthesized usingrandom primers with Thermoscript RT-PCR System (Invitrogen). PCR was runfor 20 cycles using Pfx polymerase. Primers: ACTIN-1, 5′CCATGGATGATGATATCGCC-3′ (SEQ ID NO:75); ACTIN-2,5′-TAGAAGCATTMCGGTGGAC-3′ (SEQ ID NO:76); β-catenin −1,5′-GACAATGGCTACTCAAGCTG-3′(SEQ ID NO:77); β-catenin −2,5′-CAGGTCAGTATCAAACCAGG-3′ (SEQ ID NO:78).

Western Blot.

Cells were washed twice with ice-cold phosphate-buffered saline andlysed in lysis buffer (50 mM HEPES, pH 7.5, 0.5% Nonidet P-40, 150 mMNaCl, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 1 mMdithiothreitol, 1 mM NaF, 2 mM phenylmethylsulfonyl fluoride, and 10μg/ml each of pepstatin, leupeptin, and aprotinin). 20 μg of solubleprotein was separated by SDS-PAGE and transferred to PVDF membranes.Primary Antibodies against β-catenin, Nbs1, Survivin, p21, Rsk1, k-Ras,Stat3, PCNA, NQO1, Actin (Santa Cruz), EF2, p70S6K, mTOR, PTEN (CellSignaling Technology), Ago2 (Wako), Dicer (Novas), and Parp1 (EMDBiosciences) were used in this study. The antigen-antibody complexeswere visualized by enhanced chemiluminescence (GE Biosciences).

5′-RACE Analysis

Total RNA (5 μg) from Hela cells treated with non-silencing aiRNA oraiRNA was ligated to GeneRacer™ RNA adaptor (Invitrogen,5′-CGACUGGAGCACGAGGACACUGACAUGGACUGAAGGAGUAGAAA-3′ (SEQ ID NO:79))without any prior processing. Ligated RNA was reverse transcribed intocDNA using a random primer. To detect cleavage product, PCR wasperformed using primers complementary to the RNA adaptor (GeneRacer™ 5′Nested Primer: 5′-GGACACTGACATGGACTGAAGGAGTA-3′ (SEQ ID NO:80)) andβ-catenin specific primer (GSP: 5′-CGCATGATAGCGTGTCTGGAAGCTT-3′ (SEQ IDNO:81)). Amplification fragments were resolved on 1.4% agarose gel andsized using a 1-kb Plus DNA Ladder (Invitrogen). Specific cleavage sitewas further confirmed by DNA sequencing.

Interferon-Response Detection.

For the experiment in FIG. 15a , PBMC were incubated directly with 100nM β-catenin siRNA or aiRNA. Total RNA was purified at 16 hours usingTRIZOL, and levels of interferon responsive gene expression weredetermined by RT-PCR as described by the manufacturer (SystemBiosciences). For the experiment in FIG. 15b , Hela cells were mocktransfected or transfected with 100 nM of the indicated aiRNA or siRNAfor 24 hours. Total RNA was purified using TRIZOL and levels ofinterferon responsive gene expression were determined by RT-PCR. Formicroarray analysis, Hela cells were transfected with 100 nM aiRNA orsiRNA. Total RNA was purified at 24 hours using TRIZOL, and RNA was usedfor hybridization to Human Genome U133 Plus 2.0 GeneChip (Affymetrix)according to the manufacturer's protocol (ExpressionAnalysis, Inc.). RNAfrom DharmaFECT 1 treated cells was used as control. To calculatetranscript expression values, Microarray Suite 5.0 was used withquantile normalization, and transcripts with sufficient hybridizationsignals to be called present (P) were used in this study.

aiRNA and siRNA Sequences

Sequence and structure of aiRNA and siRNA duplexes were listed in Table2. Location of point mutation is framed in the k-Ras aiRNA.

TABLE 2 siβ-catenin     G U A G C U G A U A U U G A U G G A C U USEQ ID NO: 117     ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦U U C A U C G A C U A U A A C U A C C U G SEQ ID NO: 118 aiβ-catenin      G C U G A U A U U G A U G G A SEQ ID NO: 119       ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦¦ ¦ ¦ ¦ ¦ ¦ ¦ C A U C G A C U A U A A C U A C C U G A A SEQ ID NO: 120siNbs1     A U C A U G C U G U G U U A A C U G C U U SEQ ID NO: 121    ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦U U U A G U A C G A C A C A A U U G A C G SEQ ID NO: 122 aiNbs1      A U G C U G U G U U A A C U G SEQ ID NO: 123       ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦¦ ¦ ¦ ¦ ¦ ¦ ¦ U A G U A C G A C A C A A U U G A C G A A SEQ ID NO: 124siEF2     G G C C C U C U U A U G A U G U A U A U U SEQ ID NO: 125     ¦¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦U U C C G G G A G A A U A C U A C A U A U SEQ ID NO: 126 aiEF2      C C U C U U A U G A U G U A U SEQ ID NO: 127       ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦¦ ¦ ¦ ¦ ¦ ¦ ¦ C C G G G A G A A U A C U A C A U A U A A SEQ ID NO: 128siStat3     G C C A G C A A A G A A U C A C A U G U U SEQ ID NO: 129    ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦U U C G G U C G U U U C U U A G U G U A C SEQ ID NO: 130 aiStat3      A G C A A A G A A U C A C A U SEQ ID NO: 131       ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦¦ ¦ ¦ ¦ ¦ ¦ ¦ C G G U C G U U U C U U A G U G U A C A A SEQ ID NO: 132siPTEN     A G C U A A A G G U G A A G A U A U A U U SEQ ID NO: 133    ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦U U U C G A U U U C C A C U U C U A U A U SEQ ID NO: 134 aiPTEN      U A A A G G U G A A G A U A U SEQ ID NO: 135       ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦¦ ¦ ¦ ¦ ¦ ¦ ¦ U C G A U U U C C A C U U C U A U A U A A SEQ ID NO: 136sip70S6K     C C G U G U U U G A U U U G G A U U U U U SEQ ID NO: 137    ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦U U G G C A C A A A C U A A A C C U A A A SEQ ID NO: 138 aip70S6K      U G U U U G A U U U G G A U U SEQ ID NO: 139       ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦¦ ¦ ¦ ¦ ¦ ¦ ¦ G G C A C A A A C U A A A C C U A A A A A SEQ ID NO: 140simTOR     G C A G A A U U G U C A A G G G A U A U U SEQ ID NO: 141    ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦U U C G U C U U A A C A G U U C C C U A U SEQ ID NO: 142 aimTOR      G A A U U G U C A A G G G A U SEQ ID NO: 143       ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦¦ ¦ ¦ ¦ ¦ ¦ ¦ C G U C U U A A C A G U U C C C U A U A A SEQ ID NO: 144siRsk1     G G A A A U U G G A A C A C A G U U U U U SEQ ID NO: 145    ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦U U C C U U U A A C C U U G U G U C A A A SEQ ID NO: 146 aiRsk1      A A U U G G A A C A C A G U U SEQ ID NO: 147       ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦¦ ¦ ¦ ¦ ¦ ¦ ¦ C C U U U A A C C U U G U G U C A A A A A SEQ ID NO: 148siPCNA     U G G A G A U G C U G U U G U A A U U U U SEQ ID NO: 149    ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦U U A C C U C U A C G A C A A C A U U A A SEQ ID NO: 150 aiPCNA      A G A U G C U G U U G U A A U SEQ ID NO: 151       ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦¦ ¦ ¦ ¦ ¦ ¦ ¦ A C C U C U A C G A C A A C A U U A A A A SEQ ID NO: 152siParp1     G U G G C G A A G A A G A A A U C U A U U SEQ ID NO: 153    ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦U U C A C C G C U U C U U C U U U A G A U SEQ ID NO: 154 aiParp1      G C G A A G A A G A A A U C U SEQ ID NO: 155       ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦¦ ¦ ¦ ¦ ¦ ¦ ¦ C A C C G C U U C U U C U U U A G A U A A SEQ ID NO: 156siSurvivin      A A G G A G A U C A A C A U U U U C A dTdTSEQ ID NO: 157      ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦dTdT U U C C U C U A G U U G U A A A A G U SEQ ID NO: 158 aiSurvivin       A G G A G A U C A A C A U U U SEQ ID NO: 159        ¦ ¦ ¦ ¦ ¦ ¦ ¦¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ dTdT U U C C U C U A G U U G U A A A A G USEQ ID NO: 160 siNQO1      G C C G C A G A C C U U G U G A U A U dTdTSEQ ID NO: 161      ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦dTdT C G G C G U C U G G A A C A C U A U A SEQ ID NO: 162 aiNQO1      G C A G A C C U U G U G A U A SEQ ID NO: 163       ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦¦ ¦ ¦ ¦ ¦ ¦ ¦ C G G C G U C U G G A A C A C U A U A A A SEQ ID NO: 164sip21     A G G C C C G C U C U A C A U C U U C U U SEQ ID NO: 165     ¦¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦U U U C C G G G C G A G A U G U A G A A G SEQ ID NO: 166 aip21      C C C G C U C U A C A U C U U SEQ ID NO: 167       ¦ ¦ ¦ ¦ ¦ ¦ ¦ ¦¦ ¦ ¦ ¦ ¦ ¦ ¦ U C C G G G C G A G A U G U A G A A G A A SEQ ID NO: 168aik-Ras

SEQ ID NO: 169 SEQ ID NO: 170In Life Evaluations

Daily examinations into the health status of each animal were alsoconducted. Body weights were checked every three days. Food and waterwas supplied daily according to the animal husbandry procedures of thefacility. Treatment producing >20% lethality and or >20% net body weightloss were considered toxic. Results are expressed as mean tumor volume(mm³)±SE. P Values<0.05 are considered to be statistically relevant.

Animal Husbandry

Male or female athymic nude mice 4-5 weeks (Charles River Laboratories,Wilmington, Mass.), were acclimated to the animal housing facility forat least 1 week before study initiation. All of the experimentalprocedures utilized were consistent with the guidelines outlined by theAmerican Physiology Society and the Guide for the Care and Use ofLaboratory Animals and were also approved by the Institutional AnimalCare and Use Committee of Boston Biomedical Inc. The animals were housedin groups of four in wood chip bedded cages in a room having controlledtemperature (68° F.-72° F.), light (12-h light-dark cycle), and humidity(45-55%). The animals were allowed free access to water and food duringthe experiment.

Example 1. Asymmetric Interfering RNA (aiRNA) Causes Gene-SpecificSilencing in Mammalian Cells

The siRNA structural scaffold is considered the essential configurationfor incorporating into RISC and mediating RNAi(Elbashir et al., 2001a;Elbashir et al., 2001 b; Elbashir et al., 2001c; Rana, 2007; Zamore etal., 2000). However, very little is known about RNA duplex scaffoldrequirements for RISC incorporation and gene silencing. To investigatethe structural scaffold requirements for an efficient RNAi mediator andRISC substrate, we first determined if RNA duplexes shorter than siRNAscould mediate gene silencing. The length of double stranded (ds) RNA isan important determinant of its propensity in activating protein kinaseR (PKR)-mediated non-specific interferon responses, increased synthesiscost, and delivery challenges (Elbashir et al., 2001b; Sledz et al.,2003). We designed a series of short dsRNAs ranging from 12 to 21 bpwith 2 nucleotide 3′ overhangs or blunt ends targeting differentmammalian genes. No gene silencing was detected after the length wasreduced below 19 bp (data not shown), which is consistent with previousreports in Drosophila Melanogaster cell lysate (Elbashir et al., 2001b)and the notion that 19-21 bp is the shortest siRNA duplex that mediatesRNAi (Elbashir et al., 2001a; Elbashir et al., 2001b; Elbashir et al.,2001c; Rana, 2007; Zamore et al., 2000).

We next tested if RNA duplexes of non-siRNA scaffold with an asymmetricconfiguration of overhangs can mediate gene silencing. The siRNA duplexcontains a symmetrical sense strand and an antisense strand. While theduplex siRNA structure containing a 3′ overhang is required forincorporation into the RISC complex, following Argonaute (Ago) mediatedcleavage of the sense strand, the antisense strand directs cleavage ofthe target mRNA (Hammond et al., 2001; Matranga et al., 2005; Tabara etal., 1999). We sought to make asymmetric RNA duplexes of various lengthswith overhangs at the 3′ and 5′ ends of the antisense strand.

Oligos with sequences shown in Table 3 were confirmed by 20%polyacrylamide gel after annealing. As shown in FIG. 3A, each lane wasloaded as follows: lane 1, 21nt/21nt; lane 2, 12nt (a)/21nt; lane 3,12nt (b)/21nt; lane 4, 13nt/13nt; lane 5, 13nt/21nt; lane 6, 14nt/14nt;lane 7, 14nt(a)/21nt; lane 8, 14nt(b)/21nt; lane 9, 15nt/15nt; lane 10,15nt/21nt.

TABLE 3 Oligos Sequences 21 nt/21 nt 5′-GUAGCUGAUAUUGAUGGACTT-3′(SEQ ID NO: 82) 3′-TTCAUCGACUAUAACUACCUG-5′ (SEQ ID NO: 83)12 nt/21 nt (a) 5′-UGAUAUUGAUGG-3′ (SEQ ID NO: 84)3′-CAUCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 39) 12 nt/21 nt (b)5′-CUGAUAUUGAUG-3′ (SEQ ID NO: 85) 3′-CAUCGACUAUAACUACCUGAA-5′(SEQ ID NO: 39) 13 nt/21 nt 5′-CUGAUAUUGAUGG-3′ (SEQ ID NO: 86)3′-CAUCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 39) 14 nt/21 nt (a)5′-GCUGAUAUUGAUGG-3′ (SEQ ID NO: 87) 3′-CAUCGACUAUAACUACCUGAA-5′(SEQ ID NO: 39) 14 nt/21 nt (b) 5′-CUGAUAUUGAUGGA-3′ (SEQ ID NO: 88)3′-CAUCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 39) 15 nt/21 nt5′-GCUGAUAUUGAUGGA-3′ (SEQ ID NO: 34) 3′-CAUCGACUAUAACUACCUGAA-5′(SEQ ID NO: 39)

HeLa cells were plated at 200,000 cells/well into a 6 well cultureplate. As shown in FIG. 3B, 24 hours later they were transfected withscramble siRNA (lane 1), 21-bp siRNA targeted E2F1 (lane 2, as a controlfor specificity) or 21-bp siRNA targeted beta-catenin (lane 3, as apositive control), or the same concentration of aiRNA of differentlength mix: 12nt(a)/21nt (lane 4); 12nt (b)/21nt (lane 5); 13nt/21nt(lane 6); 14nt (a)/21nt (lane 7); 14nt (b)/21nt (lane 8); 15nt/21 nt(lane 9). Cells were harvested 48 hours after transfection. Expressionof β-cantenin was determined by Western blot. E2F1 and actin are used ascontrols. The results demonstrate that asymmetric interfering RNA(aiRNA) causes gene-specific silencing in mammalian cells.

In order to determine the structural features of aiRNA important inaiRNA function, we generated multiple aiRNA oligonucleotides based onmodification of the core 15/21 dual anti-sense overhang structure (Table4). The aiRNAs, summarized in Table 4, contained modificationsincluding, but not limited to, length of the sense and anti-sensestrands, degree of sense and anti-sense overhangs, and RNA-DNA hybridoligonucleotides.

Modification to the parental 15/21 aiRNA structure was done by alteringthe sense strand, anti-sense strand, or both (Table 4). Modified aiRNAduplexes were transfected into Hela cells at 50 nM for 48 hours. Westernblots for β-catenin and actin were used to examine the degree of genesilencing compared to the parental 15/21 aiRNA and to the traditionalsiRNA structure. aiRNA modifications were also tested which containeddual sense strand overhangs. These oligonucleotides contain a 21 basesense strand paired to differing length anti-sense strands. In addition,we also examined the activity of aiRNA oligonucleotides that have beenmodified with DNA bases. DNA substitutions were done on both theanti-sense and sense strands (Table 3). RNA-DNA hybrid oligonucleotidestested contained 1 or more DNA substitutions in either the sense oranti-sense strand, or contained 21 base anti-sense RNA paired withindicated length of DNA sense strand. The gene silencing results ofthese various aiRNAs were shown in FIGS. 4 and 5.

Taken together, these data provide structural clues to aiRNA function.

Regarding the sense strand, our data indicate that the length of 15bases works well, while lengths between 14 and 19 bases remainfunctional. The sense strand can match any part of the anti-sensestrand, provided that the anti-sense overhang rules are met. Replacementof a single RNA base with DNA at either the 5′ or 3′ end of the sensestrand is tolerated and may even provide increased activity.

With respect to the anti-sense strand length, the length of 21 basesworks well, 19-22 bases retains activity, and activity is decreased whenthe length falls below 19 bases or increases above 22 bases. The 3′ endof the anti-sense strand requires an overhang of 1-5 bases with a 2-3base overhang being preferred, blunt end shows a decrease in activity.Base pairing with the target RNA sequence is preferred, and DNA basereplacement up to 3 bases is tolerated without concurrent 5′ DNA basereplacement. The 5′ end of the anti-sense strand prefers a 0-4 baseoverhang, and does not require an overhang to remain active. The 5′ endof the anti-sense strand can tolerate 2 bases not matching the targetRNA sequence, and can tolerate DNA base replacement up to 3 baseswithout concurrent 3′ DNA base replacement.

With respect to mismatched or chemically modified bases, we find thatboth mismatches and one or more chemically modified bases in either thesense or anti-sense strand is tolerated by the aiRNA structure.

TABLE 4 aiRNA sequences used for FIGS. 4-5 aiRNA # Generic StructureSequence  1 15-21 (NNN-NNN) 5′-GCUGAUAUUGAUGGA (SEQ ID NO: 34)CAUCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 39)  2 15-21a (NNNNNN---blunt)5′-GAUAUUGAUGGACUU (SEQ ID NO: 36) CAUCGACUAUAACUACCUGAA-5′(SEQ ID NO: 39)  3 15-21b (blunt---NNNNNN)5′-GUAGCUGAUAUUGAU (SEQ ID NO: 89) CAUCGACUAUAACUACCUGAA-5′(SEQ ID NO: 39)  4 15-21c (NNNN---NN) 5′-CUGAUAUUGAUGGAC (SEQ ID NO: 38)CAUCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 39)  5 15-21d (NN---NNNN)5′-AGCUGAUAUUGAUGG (SEQ ID NO: 40) CAUCGACUAUAACUACCUGAA-5′(SEQ ID NO: 39)  7 15-18b (blunt cut 3′ ---NNN)5′-GCUGAUAUUGAUGGA (SEQ ID NO: 34) CGACUAUAACUACCUGAA-5′ (SEQ ID NO: 90) 8 15-21d (N---NNNNN) 5′-UAGCUGAUAUUGAUG (SEQ ID NO: 41)CAUCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 39)  9 15-21e (NNNNN---N)5′-UGAUAUUGAUGGACU (SEQ ID NO: 42) CAUCGACUAUAACUACCUGAA-5′(SEQ ID NO: 39) 10 15-22a (NNNN---NNN)5′-GCUGAUAUUGAUGGA (SEQ ID NO: 34) UCAUCGACUAUAACUACCUGAA-5′(SEQ ID NO: 43) 11 15-22b (NNN---NNNN)5′-GCUGAUAUUGAUGGA (SEQ ID NO: 34) CAUCGACUAUAACUACCUGAAA-5′(SEQ ID NO: 35) 13 15-24a (NNNNN---NNNN)5′-GCUGAUAUUGAUGGA (SEQ ID NO: 34) UUCAUCGACUAUAACUACCUGUAA-5′(SEQ ID NO: 91) 14 15-24b (NNNN---NNNNN)5′-GCUGAUAUUGAUGGA (SEQ ID NO: 34) UCAUUCGACUAUAACUACCUGUCAA-5′(SEQ ID NO: 92) 15 15-27 (NNNNNN---NNNNNN)5′-GCUGAUAUUGAUGGA (SEQ ID NO: 34) GUUCAUCGACUAUAACUACCUGUCAUA-5′(SEQ ID NO: 93) 16 15-20a (NNN---NN) 5′-GCUGAUAUUGAUGGA (SEQ ID NO: 34)CAUCGACUAUAACUACCUGA-5′ (SEQ ID NO: 94) 17 15-20b (NNNN---N)5′-GCUGAUAUUGAUGGA (SEQ ID NO: 34) UCAUCGACUAUAACUACCUG-5′(SEQ ID NO: 95) 18 15-20c (NN-NNN) 5′-GCUGAUAUUGAUGGA (SEQ ID NO: 34)AUCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 37) 21 15-19c (NNNN-blunt)5′-GCUGAUAUUGAUGGA (SEQ ID NO: 34) UCAUCGACUAUAACUACCU-5′(SEQ ID NO: 96) 22 15-18a (NN---N) 5′-GCUGAUAUUGAUGGA (SEQ ID NO: 34)AUCGACUAUAACUACCUG-5′ (SEQ ID NO: 97) 23 15-18b (NNN-blunt)5′-GCUGAUAUUGAUGGA (SEQ ID NO: 34) CAUCGACUAUUAACUACCU-5′(SEQ ID NO: 98) 24 15-18c (blunt---NNN)5′-GCUGAUAUUGAUGGA (SEQ ID NO: 34) CGACUAUAACUACCUGAA-5′ (SEQ ID NO: 90)25 15-17a (NN---blunt) 5′-GCUGAUAUUGAUGGA (SEQ ID NO: 34)AUCGACUAUAACUACCU-5′ (SEQ ID NO: 99) 26 15-17b (blunt---NN)5′-GCUGAUAUUGAUGGA (SEQ ID NO: 34) CGACUAUAACUACCUGA-5′ (SEQ ID NO: 100)29 14-20 (NNN-NNN) 5′-GCUGAUAUUGAUGG (SEQ ID NO: 87)CAUCGACUAUAACUACCUGA-5′ (SEQ ID NO: 94) 30 14-19a (NNN---NN)5′-GCUGAUAUUGAUGG (SEQ ID NO: 87) CAUCGACUAUAACUACCUG-5′(SEQ ID NO: 101) 31 14-19b (NN---NNN) 5′-GCUGAUAUUGAUGG (SEQ ID NO: 87)AUCGACUAUAACUACCUGA-5′ (SEQ ID NO: 102) 33 14-18b (NNN---N)5′-GCUGAUAUUGAUGG (SEQ ID NO: 87) CAUCGACUAUUAACUACCU-5′ (SEQ ID NO: 98)34 16-21a (NNN---NN) 5′-GCUGAUAUUGAUGGAC (SEQ ID NO: 44)CAUCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 39) 35 16-21b (NN---NNN)5′-AGCUGAUAUUGAUGGA (SEQ ID NO: 45) CAUCGACUAUAACUACCUGAA-5′(SEQ ID NO: 39) 36 17-21 (NN---NN) 5′-AGCUGAUAUUGAUGGAC (SEQ ID NO: 46)CAUCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 39) 37 18-21a (NN---N)5′-AGCUGAUAUUGAUGGACU (SEQ ID NO: 47) CAUCGACUAUAACUACCUGAA-5′(SEQ ID NO: 39) 38 18-21b (N---NN) 5′-UAGCUGAUAUUGAUGGAC (SEQ ID NO: 48)CAUCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 39) 39 18-21c (NNN---blunt)5′-GCUGAUAUUGAUGGACUU (SEQ ID NO: 49) CAUCGACUAUAACUACCUGAA-5′(SEQ ID NO: 39) 40 19-21a (NN---blunt)5′-AGCUGAUAUUGAUGGACUU (SEQ ID NO: 50) CAUCGACUAUAACUACCUGAA-5′(SEQ ID NO: 39) 41 18-21b (blunt---NNN)5′-GUAGCUGAUAUUGAUGGA (SEQ ID NO: 103) CAUCGUCUAUAACUACCUGAA-5′(SEQ ID NO: 104) 42 19-21c (N---N)5′-UAGCUGAUAUUGAUGGACU (SEQ ID NO: 51) CAUCGACUAUAACUACCUGAA-5′(SEQ ID NO: 39) 43 20-21a (N---blunt)5′-UAGCUGAUAUUGAUGGACUU (SEQ ID NO: 52) CAUCGACUAUAACUACCUGAA-5′(SEQ ID NO: 39) 44 20-21b (blunt---N)5′-GUAGCUGAUAUUGAUGGACU (SEQ ID NO: 53) CAUCGACUAUAACUACCUGAA-5′(SEQ ID NO: 39) 45 Mismatch and miRNA 5′-GCUGAUAUUGAAGGA (SEQ ID NO: 54)CAUCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 39) 46 5′ end homologous to target5′-GCUGAUAUUGAUGGA (SEQ ID NO: 34) CAUCGACUAUAACUACCUGUC-5′(SEQ ID NO: 55) 47 NNNNNNNNNNNNNNN (SEQ ID NO: 108)5′-GCUGAUAUUGAUGGA (SEQ ID NO: 34) 3′NNNNNNNNNNNNNNNNNNDDD-5′CAUCGACUAUAACUACCUgaa-5′ (SEQ ID NO: 56) (SEQ ID NO: 109) 48NNNNNNNNNNNNNNN (SEQ ID NO: 108) 5′-GCUGAUAUUGAUGGA (SEQ ID NO: 34)3′DDDNNNNNNNNNNNNNNNNNN-5′ catCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 57) 49NNNNNNNNNNNNNNN (SEQ ID NO: 108) 5′-GCUGAUAUUGAUGGA (SEQ ID NO: 34)3′DDDNNNNNNNNNNNNNNNDDD-5′ catCGACUAUAACUACCUgaa-5′ (SEQ ID NO: 105)(SEQ ID NO: 110) 51 DNDNNNNNNNDNDND (SEQ ID NO: 116)5′-gCTGAUAUUGaUgGa (SEQ ID NO: 106) 3′NNNNNNNNNNNNNNNNNNNNN-5′CAUCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 39) (SEQ ID NO: 111) 52DNNNNNNNNNNNNNN (SEQ ID NO: 114) 5′-gCUGAUAUUGAUGGA (SEQ ID NO: 58)3′NNNNNNNNNNNNNNNNNNNNN-5′ CAUCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 39)(SEQ ID NO: 111) 53 NNNNNNNNNNNNNND (SEQ ID NO: 115)5′-GCUGAUAUUGAUGGa (SEQ ID NO: 59) 3′NNNNNNNNNNNNNNNNNNNNN-5′CAUCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 39) (SEQ ID NO: 113) 545′-UAGCUGAUAUUGAUG (SEQ ID NO: 41) UUCAUCGACUAUAACUACCUG-5′(SEQ ID NO: 107) 55 5′-GUAGCUGAUAUUGAUGGA (SEQ ID NO: 103)UUCAUCGACUAUAACUACCUG-5′ (SEQ ID NO: 107) 565′-AGCUGAUAUUGAUGGA (SEQ ID NO: 45) UUCAUCGACUAUAACUACCUG-5′(SEQ ID NO: 107) 57 DNNNNNNNNNNNNNN (SEQ ID NO: 114)5′-gCUGAUAUUGAUGGA (SEQ ID NO: 58) 3′DDDNNNNNNNNNNNNNNNNNN-5′catCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 57) (SEQ ID NO: 112) 58DNNNNNNNNNNNNNN (SEQ ID NO: 114) 5′-gCUGAUAUUGAUGGA (SEQ ID NO: 58)3′NNNNNNNNNNNNNNNNNNDDD-5′ CAUCGACUAUAACUACCUgaa-5′ (SEQ ID NO: 56)(SEQ ID NO: 109) 59 NNNNNNNNNNNNNND (SEQ ID NO: 115)5′-GCUGAUAUUGAUGGa (SEQ ID NO: 59) 3′DDDNNNNNNNNNNNNNNNNNN-5′catCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 57) (SEQ ID NO: 112) 60NNNNNNNNNNNNNND (SEQ ID NO: 115) 5′-GCUGAUAUUGAUGGa (SEQ ID NO: 59)3′NNNNNNNNNNNNNNNNNNDDD-5′ CAUCGACUAUAACUACCUgaa-5′ (SEQ ID NO: 56)(SEQ ID NO: 109) 61 NNNNNNNNNNNNNNNNNNN5′-UAGCUGAUAUUGAUGGACU (SEQ ID NO: 51) (SEQ ID NO: 111)catCGACUAUAACUACCUGAA-5′ (SEQ ID NO: 57) 3′DDDNNNNNNNNNNNNNNNNNN-5′(SEQ ID NO: 112) 62 NNNNNNNNNNNNNNNNNNN5′-UAGCUGAUAUUGAUGGACU (SEQ ID NO: 51) (SEQ ID NO: 111)CAUCGACUAUAACUACCUgaa-5′ (SEQ ID NO: 56) 3′NNNNNNNNNNNNNNNNNNDDD-5′(SEQ ID NO: 109)In table 4, A, U, G, C represent nucleotides, while a, t, g, c representdeoxynucleotides.

Example 2. Mechanism of Gene Silencing Triggered by aiRNA

To investigated the mechanism of gene knockdown induced by aiRNA, wefirst determined if the gene silencing by aiRNA occurs at translationalor mRNA level. Northern blot analysis of β-catenin in cells transfectedwith 10 nM of the 15 bp aiRNA showed that the aiRNA reduced mRNA levelsby over 95% within 24 hours and the decrease lasted more than 4 days(FIG. 6a ), suggesting that aiRNA mediates gene silencing at the mRNAlevel. The reduction of β-catenin mRNA induced by aiRNA wassubstantially more rapid, efficacious and durable than by siRNA (FIG. 6a). We further determined if the 15 bp aiRNA catalyzed the site-specificcleavage of β-catenin mRNA. Total RNA isolated from cells transfectedwith the 15 bp aiRNA was examined by rapid amplification of cDNA ends(5′-RACE) and PCR for the presence of the β-catenin mRNA cleavagefragments (FIG. 6b ). We detected β-catenin cleavage fragments at 4 and8 hours following aiRNA transfection (FIG. 6c ). Sequence analysisshowed that cleavage was taking place within the aiRNA target sequencebetween bases 10 and 11 relative to the 5′ end of the aiRNA antisensestrand (FIG. 6d ). No such cleavage fragments were observed followingtransfection with a scrambled aiRNA (FIG. 6c ). These resultsdemonstrate that aiRNA induced potent and efficacious gene silencingthrough sequence-specific cleavage of the target mRNA.

We next determined whether the novel asymmetric scaffold of aiRNA can beincorporated into the RISC. RNAi is catalyzed by RISC enzyme complexwith an Argonaute protein (Ago) as the catalytic unit of the complex(Liu et al., 2004; Matranga et al., 2005). To determine if aiRNA isincorporated into the Ago/RISC complex, we immunoprecipitated myc-taggedhuman Ago1 from cells expressing myc-tagged Ago1 (Siolas et al., 2005)after cells were transfected with aiRNA. Small RNAs associated with theRISC complex were detected by northern blotting of Agoimmunoprecipitates. Northern blot analysis revealed that the aiRNAentered the RISC complex with high efficiency (FIG. 6e ). These datasuggest the asymmetric scaffold of aiRNA can be efficiently incorporatedinto RISC.

Since aiRNA induced more efficient gene silencing than siRNA, we testedif aiRNA can give rise to RISC complex more efficiently than siRNA. Asshown in FIG. 6e , aiRNA-Ago2/RISC complexes formed faster and moreefficient than the siRNA-Ago2/RISC complexes, with more aiRNA containedin the RISC complex than the corresponding siRNA (FIG. 6e and FIG. 7A).Of note, siRNA displayed a typical pattern (21) that is consistent withformation of secondary structures by siRNA (FIG. 6e and FIG. 7). Incontrast, aiRNA displayed a single band, suggesting that the shorterlength of aiRNA may reduce or eliminate the secondary structureformation as occurred with siRNA.

Further, the asymmetric configuration of aiRNA may facilitate theformation of active RISC with antisense strand and reduce theineffective RISC formed with the sense strand (Ref. 16). Our data provedthis is true as shown in FIG. 7B, no sense strand can be detected in theRISC complex. FIG. 8A also demonstrates that while the anti-sense strandof the aiRNA strongly associates with Ago 2, the sense-strand does not.In contrast, both the anti-sense and sense strand of the siRNA associatewith Ago 2. These data suggest that aiRNA has higher efficiency informing RISC than siRNA in cells, which may underlie the superior genesilencing efficiency of aiRNA.

In addition, it has been shown that the sense strand of siRNA isrequired to be cleaved in order to be functional. Therefore, we testedif the same requirement is true for aiRNA. To do that, the nucleotide atposition 8 or 9 of the aiRNA sense strand was modified with 2′-O-methylto make it uncleavable. Our results show that the aiRNAs with theuncleavable sense strand are still functional (FIG. 8B), demonstratingaiRNA is quite different than siRNA in terms of their mechanism.

Further we asked if there is any different loading pocket for aiRNA andsiRNA. We used cold aiRNA or siRNA to compete with the radioactivelylabelled siRNA or aiRNA for the RISC complex (FIG. 9). Surprisingly, theresults show that cold aiRNA does not compete with the siRNA for RISCcomplex (FIG. 9B) and cold siRNA does not compete with aiRNA for theRISC complex either (FIG. 9C). These data indicate that aiRNA and siRNAmay load to different pockets of RISC complex.

Together, the data above suggest that aiRNA represents the firstnon-siRNA scaffold that is incorporated into RISC, providing a novelstructural scaffold that interacts with RISC. The difference of the RISCloading of aiRNA and siRNA is illustrated in our model shown in FIG. 10.Briefly, because of the asymmetric property, only the anti-sense strandis selected to stay in the RISC complex and results in a 100% efficiencyin strand selection. In contrast, siRNA is structurally symmetric. Bothanti-sense strand and sense strand of the siRNA has a chance to beselected to stay in the RISC complex and therefore siRNA has aninefficient strand selection and at the same time may cause non-specificgene silencing due to the sense strand RISC complex.

Example 3. aiRNA Mediates a More Rapid, Potent, Efficacious, and DurableGene Silencing than siRNA

To compare aiRNA with siRNA in gene silencing properties, we firstdetermined the optimal aiRNA structure for gene silencing.

The siRNA duplex contains a symmetrical sense strand and an antisensestrand. While the duplex siRNA structure containing a 3′ overhang isrequired for incorporation into the RISC complex, following Argonaute(Ago) mediated cleavage of the sense strand, the antisense stranddirects cleavage of the target mRNA (Hammond et al., 2001; Matranga etal., 2005; Tabara et al., 1999). We sought to make asymmetric RNAduplexes of various lengths with overhangs at the 3′ and 5′ ends of theantisense strand. We designed one set of such asymmetrical RNA duplexesof 12 to 15 bp with 3′ and 5′ antisense overhangs to target β-catenin(FIG. 11A), an endogenous gene implicated in cancer and stem cells(Clevers, 2006). An optimized siRNA of the standard configuration hasbeen designed to target β-catenin for triggering RNAi (Xiang et al.,2006). All aiRNAs against β-catenin were designed within the samesequence targeted by the siRNA (FIG. 11A). The results showed that theoptimal gene silencing achieved was with the 15 bp aiRNA (FIG. 11B).Therefore, we used 15 bp aiRNA to be compared with 21-mer siRNA duplexin the subsequent experiments.

To our surprise, we found that aiRNA induced potent and highlyefficacious reduction of β-catenin protein while sparing thenon-targeted control genes actin (FIG. 11C).

We next examined the onset of gene silencing by aiRNA and siRNAtargeting β-catenin. The sequence of the aiRNA and siRNA used is shownin FIG. 11A. As shown in FIG. 12, aiRNA has a more rapid onset (FIGS.12C and D) and also a better efficacy (FIGS. 12B and D).

We also compared the gene silencing effects of aiRNA and siRNA onvarious targets and multiple human cell lines. The aiRNAs were designedto target genes of different functional categories including Stat3 (FIG.13b ), NQO1 (FIG. 12d ), elongation factor 2 (EF2) (FIG. 13c ), Nbs1(FIG. 14b ), Survivin (FIG. 14b ), Parp1 (FIG. 14b ), p21 (FIG. 14b ),Rsk1 (FIG. 14c ), PCNA (FIG. 14c ), p70S6K (FIG. 14c ), mTOR (FIG. 14c), and PTEN (FIG. 14c ), besides β-catenin (FIG. 13a ) at the samesequences that have been targeted with siRNA with low efficiency (Rogoffet al., 2004). As shown in FIGS. 13 and 14, aiRNA is more efficaciousthan siRNA in silencing Stat3, β-catenin, Rsk1, p70S6K, Nbs1, mTOR, andEF2, and is as efficacious as siRNA in silencing NQO1, PCNA, Survivin,PTEN, Parp1, and p21. Since the target sequences were chosen based onthe optimization for siRNA, it is possible that the efficacy and potencyof aiRNA can be further increased by targeting sites that are optimizedfor aiRNA. In addition, our data also shows that aiRNA is moreefficacious than siRNA against b-catenin in multiple cell linesincluding Hela (FIG. 13a ), H1299 (FIG. 14a , left panel) and Dld1 (FIG.14a , right panel).

Taken together, these data demonstrate that aiRNA is more efficacious,potent, rapid-onset, and durable than siRNA in mediating gene silencingin mammalian cells.

Example 4. Specificity of Gene Silencing Mediated by aiRNA

We next investigated the specificity of gene silencing mediated byaiRNAs. We first analyzed aiRNAs that target the wildtype k-Ras allele.DLD1 cells contain wild-type k-Ras while SW480 cells contain mutantk-Ras that has a single base pair substitution (FIG. 14d ). Transfectionof DLD1 cells with aiRNA targeting wildtype k-Ras showed effectivesilencing, but no silencing of mutant k-Ras was observed in the SW480cells. These data demonstrate that aiRNA mediates allele specific genesilencing.

The activation of an interferon-like response is a major non-specificmechanism of gene silencing. A primary reason that siRNAs are used forgene silencing is that the dsRNA of shorter than 30 bp has reducedability to activate the interferon-like response in mammalian cells(Bernstein et al., 2001; Martinez and Tuschl, 2004; Sledz et al., 2003).We tested if aiRNA showed any signs of activating the interferon-likeresponse in mammalian cells. RNA collected from PBMC cells transfectedwith aiRNA against β-catenin and Hela cells transfected with aiRNAagainst EF2 or Survivin was analyzed by RT-PCR for interferon induciblegenes. We found that aiRNA transfection showed no increase by RT-PCR ofany of the interferon inducible genes tested, while levels of targetedmRNAs were reduced relative to control transfected cells (FIGS. 15a andb ). Microarray analysis was also performed to compare the changes inthe expression of known interferon response related genes induced byaiRNA and miRNA. As shown in FIG. 15c , much less changes were observedfor aiRNA compared to siRNA.

In addition, as mentioned above, sense strand-RISC complex may causenon-specific gene silencing. To compare aiRNA and siRNA on thenon-specific gene silencing mediated by sense-strand-RISC complex, cellswere co-transfected with aiRNA or siRNA and either a plasmid expressingStat3 (sense RNA) or a plasmid expressing antisense Stat3 (antisenseRNA). Cells were harvested and RNA collected at 24 hours posttransfection and relative levels of Stat3 sense or antisense RNA weredetermined by quantitative real time PCR or RT-PCR (inserts). Theresults show that aiRNA has no effect on the antisense Stat3 mRNAs whilesiRNA does (FIG. 15d ). This result demonstrate aiRNA completely abolishthe undesired non-specific gene-silencing mediated by the sensestrand-RISC complex.

In summary, we have shown that aiRNA is a novel class of gene-silencinginducers, the non-siRNA type and the smallest structural scaffold forRISC substrates and RNAi mediators (FIG. 15f ). Our data suggest thataiRNA works through RISC, the cellular RNAi machinery. Afterincorporation into RISC, aiRNA mediates sequence-specific cleavage ofthe mRNA between base 10 and 11 relative to the 5′ end of the aiRNAantisense strand. The asymmetrical configuration of aiRNA can interactmore efficiently with RISC than siRNA. Consistent with high RISC bindingefficiency, aiRNA is more potent, efficacious, rapid-onset, and durablethan siRNA in mediating gene-specific silencing against genes tested inour study. While previous studies have proposed a role of Dicer infacilitating efficient RISC formation, our data suggest aiRNA can giverise to active RISC complexes with high efficiency independent ofDicer-mediated processing.

The key feature of this novel RNA duplex scaffold is antisense overhangsat the 3′ and 5′ ends. The 12-15 bp aiRNA are the shortest RNA duplexknown to induce RNAi. While long dsRNAs triggered potent gene silencingin C. elegans and Drosophila melanogaster, gene-specific silencing inmammalian cells was not possible until siRNA duplexes were used. ThesiRNA scaffold, as defined by Dicer digestion, is characterized bysymmetry in strand lengths of 19-21 bp and 3′ overhangs (Bernstein etal., 2001), which has been considered the essential structure forincorporating into RISC to mediate RNAi. Therefore, optimization effortsfor RNAi inducers have been focused on siRNA precursors, which areinvariably larger than siRNA (Soutschek et al., 2004; Zhang and Farwell,2007). Our data suggest that siRNA is not the essential scaffold forincorporating into RISC to mediate RNAi. The aiRNAs of different lengthsdisplayed a spectrum of gene silencing efficacy and RISC incorporationefficiency, offering unique opportunity for understanding the mechanismof RISC incorporation and activation. Research is needed to furtherunderstand the structure-activity relationship of aiRNAs in RISCincorporation and RNAi induction, which should help establish a rationalbasis for optimizing aiRNAs with regards to target sequence selection,length, structure, chemical composition and modifications for variousRNAi applications.

Example 5. aiRNA is More Efficacious than siRNA In Vivo

To investigate if aiRNA is efficacious in vivo and to compare it withsiRNA, we tested the effects of aiRNA and siRNA in human colon cancerxenograft models.

Human Colon Cancer is the second leading cause of cancer death in theU.S. The Wnt β-cantenin signaling pathway is tightly regulated and hasimportant functions in development, tissue homeostasis, andregeneration. Deregulation of Wnt/P-catenin signaling is frequentlyfound in various human cancers. Eighty percent of colorectal cancersalone reveal activation of this pathway by either inactivation of thetumor-suppressor gene adenomatous polyposis coli or mutation of theproto-oncogene β-catenin.

Activation of Wnt/β-catenin signaling has been found to be important forboth initiation and progression of cancers of different tissues.Therefore, targeted inhibition of Wnt/β-catenin signaling is a rationaland promising new approach for the therapy of cancers of variousorigins.

In vitro, by ribozyme-targeting we have demonstrated the reduction ofβ-cantenin expression in human colon cancer SW480 cells and associatedinduction of cell death, indicating that β-cantenin expression israte-limiting for tumor growth in vitro.

SW480 human colon cancer cells were inoculated subcutaneously intofemale athymic nude mice (8×10⁶ cells/mouse) and allowed to formpalpable tumors. In this study, dosing began when the tumors reachedapproximately 120 mm³. Animals were treated intravenously (iv) with 0.6nmol PEI-complexed β-cantenin siRNAs, PEI-complexed β-catenin aiRNAs ora PEI-complexed unrelated siRNA as a negative control daily. The animalsreceived a total of 10 doses of siRNA, aiRNA or control. Tumors weremeasured throughout treatment. As shown in FIG. 16, intravenoustreatment with siRNA and aiRNA as a monotherapy at 0.6 nmol mg/kgsignificantly inhibited tumor growth. The % T/C value of siRNA wascalculated to be 48.8% with a p value of 0.0286. The treatment with theβ-catenin-specific aiRNAs, however, resulted in a much more potentreduction in tumor growth. The % T/C value was calculated to be 9.9%with a p value of 0.0024. There was no significant change in body weightdue to iv administration of the siRNA, aiRNA or control. These datasuggest that the systemic in vivo application of aiRNAs through PEIcomplexation upon targeting of the β-catenin offers an avenue for thedevelopment of highly efficient, specific and safe agents fortherapeutic applications for patients with colon cancer.

In addition, we also tested the effects of aiRNA and siRNA in HT29 humancolon cancer xenograft model. HT29 human colon cancer cells wereinoculated subcutaneously into female athymic nude mice (6×10⁶cells/mouse) and allowed to form palpable tumors. In this study, dosingbegan when the tumors reached approximately 200 mm³. Animals weretreated intravenously (iv) with 0.6 nmol PEI-complexed β-catenin siRNAs,PEI-complexed β-cantenin aiRNAs or a PEI-complexed unrelated siRNA as anegative control every other day. The animals received a total of 8doses of siRNA, aiRNA or control. Tumors were measured throughouttreatment. As shown in FIG. 17, intravenous treatment with siRNA andaiRNA as a monotherapy at 0.6 nmol mg/kg significantly inhibited tumorgrowth. The % T/C value of siRNA was calculated to be 78% with a p valueof 0.21. Again, the treatment with the β-catenin-specific aiRNAsresulted in an even more potent reduction in tumor growth. The % T/Cvalue was calculated to be 41% with a p value of 0.016. There was nosignificant change in body weight due to iv administration of the siRNA,aiRNA or control. These data second that the systemic in vivoapplication of aiRNAs through PEI complexation upon targeting of theβ-cantenin offers an avenue for the development of highly efficient,specific and safe agents for therapeutic applications for patients withcolon cancer.

Together, the aiRNA may significantly improve broad RNAi applications.The siRNA-based therapeutics have met with challenges, including limitedefficacy, delivery difficulty, interferon-like responses and manufacturecost (de Fougerolles et al., 2007; Iorns et al., 2007; Rana, 2007). Theimproved efficacy, potency, durability, and smaller size of aiRNAs mayhelp or overcome these challenges since aiRNA is smaller and may needless material for its delivery. Therefore, aiRNA represents new andsmallest RNA duplexes that enter RISC and mediates gene silencing ofbetter efficacy, potency, onset of action, and durability than siRNA inmammalian cells, holding significant potential for broad RNAiapplications in gene function study and RNAi-based therapies.

Other embodiments are within the following claims. While severalembodiments have been shown and described, various modifications may bemade without departing from the spirit and scope of the presentinvention. Sequence listings and related materials in the ASCII textfile named “2019-03-GHI-012_ST25.txt” and created on Mar. 22, 2019 witha size of about 38 kilobytes, is hereby incorporated by reference.

REFERENCES

-   Bernstein, E., Caudy, A. A., Hammond, S. M., and Hannon, G. J.    (2001). Role for a bidentate ribonuclease in the initiation step of    RNA interference. Nature 409, 363-366.-   Chiu, Y. L., and Rana, T. M. (2003). siRNA function in RNAi: a    chemical modification analysis. RNA (New York, N.Y. 9, 1034-1048.-   Clevers, H. (2006). Wnt/beta-catenin signaling in development and    disease. Cell 127, 469-480.-   Czauderna, F., Fechtner, M., Dames, S., Aygun, H., Klippel, A.,    Pronk, G. J., Giese, K., and Kaufmann, J. (2003). Structural    variations and stabilising modifications of synthetic siRNAs in    mammalian cells. Nucleic acids research 31, 2705-2716.-   Czech, M. P. (2006). MicroRNAs as therapeutic targets. The New    England journal of medicine 354, 1194-1195.-   de Fougerolles, A., Vomlocher, H. P., Maraganore, J., and    Lieberman, J. (2007). Interfering with disease: a progress report on    siRNA-based therapeutics. Nat Rev Drug Discov 6, 443-453.-   Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983). Accurate    transcription initiation by RNA polymerase II in a soluble extract    from isolated mammalian nuclei. Nucleic acids research 11,    1475-1489.-   Donze, O., and Picard, D. (2002). RNA interference in mammalian    cells using siRNAs synthesized with T7 RNA polymerase. Nucleic Acids    Res 30, e46.-   Dykxhoorn, D. M., Novina, C. D., and Sharp, P. A. (2003). Killing    the messenger: short RNAs that silence gene expression. Nat Rev Mol    Cell Biol 4, 457-467.-   Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K.,    and Tuschl, T. (2001a). Duplexes of 21-nucleotide RNAs mediate RNA    interference in cultured mammalian cells. Nature 411, 494-498.-   Elbashir, S. M., Lendeckel, W., and Tuschl, T. (2001b). RNA    interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev    15, 188-200.-   Elbashir, S. M., Martinez, J., Patkaniowska, A., Lendeckel, W., and    Tuschl, T. (2001c). Functional anatomy of siRNAs for mediating    efficient RNAi in Drosophila melanogaster embryo lysate. Embo J 20,    6877-6888.-   Eulalio, A., Huntzinger, E., and Izaurralde, E. (2008). Getting to    the root of miRNA-mediated gene silencing. Cell 132, 9-14.-   Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E.,    and Mello, C. C. (1998). Potent and specific genetic interference by    double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811.-   Hammond, S. M., Bernstein, E., Beach, D., and Hannon, G. J. (2000).    An RNA-directed nuclease mediates post-transcriptional gene    silencing in Drosophila cells. Nature 404, 293-296.-   Hammond, S. M., Boettcher, S., Caudy, A. A., Kobayashi, R., and    Hannon, G. J. (2001). Argonaute2, a link between genetic and    biochemical analyses of RNAi. Science 293, 1146-1150.-   Iorns, E., Lord, C. J., Turner, N., and Ashworth, A. (2007).    Utilizing RNA interference to enhance cancer drug discovery. Nature    reviews 6, 556-568.-   Kim, D. H., Behlke, M. A., Rose, S. D., Chang, M. S., Choi, S., and    Rossi, J. J. (2005). Synthetic dsRNA Dicer substrates enhance RNAi    potency and efficacy. Nat Biotechnol 23, 222-226.-   Kim, D. H., and Rossi, J. J. (2007). Strategies for silencing human    disease using RNA interference. Nature reviews 8, 173-184.-   Liu, J., Carmell, M. A., Rivas, F. V., Marsden, C. G., Thomson, J.    M., Song, J. J., Hammond, S. M., Joshua-Tor, L., and Hannon, G. J.    (2004). Argonaute2 is the catalytic engine of mammalian RNAi.    Science 305, 1437-1441.-   Mack, G. S. (2007). MicroRNA gets down to business. Nature    biotechnology 25, 631-638.-   Martinez, J., and Tuschl, T. (2004). RISC is a 5′    phosphomonoester-producing RNA endonuclease. Genes Dev 18, 975-980.-   Matranga, C., Tomari, Y., Shin, C., Bartel, D. P., and Zamore, P. D.    (2005). Passenger-strand cleavage facilitates assembly of siRNA into    Ago2-containing RNAi enzyme complexes. Cell 123, 607-620.-   Paddison, P. J., Caudy, A. A., Bernstein, E., Hannon, G. J., and    Conklin, D. S. (2002). Short hairpin RNAs (shRNAs) induce    sequence-specific silencing in mammalian cells. Genes Dev 16,    948-958.-   Patzel, V. (2007). In silico selection of active siRNA. Drug    discovery today 12, 139-148.-   Rana, T. M. (2007). Illuminating the silence: understanding the    structure and function of small RNAs. Nat Rev Mol Cell Biol 8,    23-36.-   Rogoff, H. A., Pickering, M. T., Frame, F. M., Debatis, M. E.,    Sanchez, Y., Jones, S., and Kowalik, T. F. (2004). Apoptosis    associated with deregulated E2F activity is dependent on E2F1 and    Atm/Nbs1/Chk2. Mol Cell Biol 24, 2968-2977.-   Siolas, D., Lerner, C., Burchard, J., Ge, W., Linsley, P. S.,    Paddison, P. J., Hannon, G. J., and Cleary, M. A. (2005). Synthetic    shRNAs as potent RNAi triggers. Nat Biotechnol 23, 227-231.-   Sledz, C. A., Holko, M., de Veer, M. J., Silverman, R. H., and    Williams, B. R. (2003). Activation of the interferon system by    short-interfering RNAs. Nat Cell Biol 5, 834-839.-   Soutschek, J., Akinc, A., Bramlage, B., Charisse, K., Constien, R.,    Donoghue, M., Elbashir, S., Geick, A., Hadwiger, P., Harborth, J.,    el al. (2004). Therapeutic silencing of an endogenous gene by    systemic administration of modified siRNAs. Nature 432, 173-178.-   Tabara, H., Sarkissian, M., Kelly, W. G., Fleenor, J., Grishok, A.,    Timmons, L., Fire, A., and Mello, C. C. (1999). The rde-1 gene, RNA    interference, and transposon silencing in C. elegans. Cell 99,    123-132.-   Xiang, S., Fruehauf, J., and Li, C. J. (2006). Short hairpin    RNA-expressing bacteria elicit RNA interference in mammals. Nature    biotechnology 24, 697-702.-   Zamore, P. D., and Aronin, N. (2003). siRNAs knock down hepatitis.    Nature medicine 9, 266-267.-   Zamore, P. D., Tuschl, T., Sharp, P. A., and Bartel, D. P. (2000).    RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA    at 21 to 23 nucleotide intervals. Cell 101, 25-33.-   Zhang, B., and Farwell, M. A. (2007). microRNAs: a new emerging    class of players for disease diagnostics and gene therapy. J Cell    Mol Med.-   Zhang, N. Y., Du, Q., Wahlestedt, C., and Liang, Z. (2006). RNA    Interference with chemically modified siRNA. Current topics in    medicinal chemistry 6, 893-900.

What is claimed is:
 1. An asymmetric interfering RNA duplex molecule,comprising an antisense strand and a sense strand, wherein the antisensestrand is longer than the sense strand, consists of 19, 20, 21, 22 or 23nucleotides and includes a 3′-overhang of 1, 2, 3, 4, 5, 6, 7, 8 or 9nucleotides and a 5′-overhang of 0, 1, 2, 3, 4, 5, 6, 7 or 8 nucleotideswhen duplexed with the sense strand; wherein the sense strand consistsof 12, 13, 14, 15, 16, 17, 18, or 19 nucleotides and forms adouble-stranded region with the antisense strand, and at least the firstnucleotide and last nucleotide of the sense strand base pair withnucleotides of the antisense strand, and wherein the double-strandedregion has a length of 11, 12, 13, 14, 15, 16, 17, 18, or 19 base pairs;and wherein the antisense strand is at least 70% complementary to atarget mRNA and has the last nucleotide of its 3′ end consisting of anA, U, G or C ribonucleotide.
 2. The RNA duplex molecule of claim 1,wherein the sense strand of the RNA duplex does not substantiallymediate off-target silencing.
 3. The RNA duplex molecule of claim 1,wherein the RNA duplex is more effective at silencing the expressednucleotide sequence of the target gene than a corresponding 21-mer siRNAduplex targeting the same expressed nucleotide sequence of the targetgene.
 4. The RNA duplex molecule of claim 1, wherein the RNA duplex doesnot induce an interferon response.
 5. The RNA duplex molecule of claim1, wherein the double-stranded region has a length of 12, 13, 14, 15, 16or 17 base pairs.
 6. The RNA duplex molecule of claim 1, wherein thedouble-stranded region consists of perfectly complementary sequences. 7.The RNA duplex molecule of claim 1, wherein the double-stranded regioncomprises at least one nick, gap or mismatch.
 8. The RNA duplex moleculeof claim 1, wherein the sense strand consists of 14 or 15 nucleotides.9. The RNA duplex molecule of claim 1, wherein the antisense strandconsists of 21 nucleotides.
 10. The RNA duplex molecule of claim 1,wherein the GC content of the double stranded region is 30%-50%.
 11. TheRNA duplex molecule of claim 1, wherein the antisense strand comprises a3′-overhang of 2, 3, 4, 5 or 6 nucleotides.
 12. The RNA duplex moleculeof claim 1, wherein the antisense strand comprises a 5′-overhang of 0,1, 2, 3, 4 or 5 nucleotides.
 13. The RNA duplex molecule of claim 1,wherein the antisense strand comprises a 5′-overhang of 2, 3 or 4nucleotides and a 3′-overhang of 2, 3 or 4 nucleotides.
 14. The RNAduplex molecule of claim 1, wherein the antisense strand comprises a5′-overhang of 3 nucleotides and a 3′-overhang of 3 nucleotides.
 15. TheRNA duplex molecule of claim 1, wherein the antisense strand comprises a3′-overhang of 1, 2, 3, 4, 5, 6, 7, 8 or 9 nucleotides and a 5′-bluntend.
 16. The RNA duplex molecule of claim 1, wherein the antisensestrand comprises a 3′-overhang of 6 nucleotides and a 5′-blunt end. 17.The RNA duplex molecule of claim 1, wherein at least one nucleotide ofthe 5′ end of the antisense strand is selected from the group consistingof A, U, and dT.
 18. The RNA duplex molecule of claim 1, wherein the5′-end of the antisense oligonucleotide comprises an “AA”, “UU” or“dTdT” motif.
 19. The RNA duplex molecule of claim 1, wherein the3′-overhang and/or 5′-overhang is stabilized against degradation eitherthrough chemical modification or secondary structure.
 20. The RNA duplexmolecule of claim 1, wherein the RNA duplex molecule contains at leastone modified nucleotide or its analogue.
 21. The RNA duplex molecule ofclaim 20, wherein the at least one modified nucleotide or its analogueis sugar-, backbone-, and/or base-modified ribonucleotide.
 22. The RNAduplex molecule of claim 21, wherein the backbone-modifiedribonucleotide has a modification in a phosphodiester linkage withanother ribonucleotide.
 23. The RNA duplex molecule of claim 22, whereinthe phosphodiester linkage is modified to include at least one of anitrogen or sulphur heteroatom.
 24. The RNA duplex molecule of claim 20,wherein the at least one modified nucleotide or its analogue is anunusual base or a modified base.
 25. The RNA duplex molecule of claim20, wherein the at least one modified nucleotide or its analoguecomprises inosine or a tritylated base.
 26. The RNA duplex molecule ofclaim 20, wherein the nucleotide analogue is a sugar-modifiedribonucleotide, wherein the 2′—OH group is replaced by a group selectedfrom H, OR, R, halo, SH, SR, NH2, NHR, NR2 or CN, wherein each R isindependently C1-C6 alkyl, alkenyl or alkynyl, and halo is F, Cl, Br orI.
 27. The RNA duplex molecule of claim 26, wherein the 2′-OH isreplaced by a 2′-O-methyl group and/or 2′-F.
 28. The RNA duplex moleculeof claim 20, wherein the nucleotide analogue is a backbone-modifiedribonucleotide containing a phosphothioate group.
 29. The RNA duplexmolecule of claim 1, wherein the antisense strand comprises at least onedeoxynucleotide.
 30. The RNA molecule of claim 1, wherein at least onenucleotide of the 5′-end of antisense strand is not complementary to thetarget mRNA sequence.
 31. The RNA molecule of claim 1, wherein theantisense strand and sense strand are joined by a chemical linker. 32.The RNA duplex molecule of claim 1 being conjugated to an entityselected from the group consisting of peptide, antibody, polymer, lipid,oligonucleotide, cholesterol, and aptamer.
 33. A composition comprisingthe RNA duplex molecule of claim 1 and a pharmaceutically acceptableexcipient, carrier, or diluent selected from the group consisting of apharmaceutical carrier, a positive-charge carrier, a liposome, a proteincarrier, a polymer, a nanoparticle, a nanoemulsion, a lipid, and alipoid.
 34. A method of treating or preventing a cancer or tumorcomprising administering to a subject in need thereof the RNA duplexmolecule of claim
 1. 35. The method of claim 34, wherein the RNA duplexmolecule is administered via a route selected from the group consistingof injection, oral administration, inhalation, topical, and regionaladministration.
 36. The method of claim 34, wherein the cancer is coloncancer.